Methods for preparing antibody drug conjugates

ABSTRACT

The subject matter described herein is directed to methods of preparing certain antibody-drug conjugates (ADCs) wherein the antibody is linked to the drug through a linker, wherein the drug contains a heteroaryl group having a secondary nitrogen, and the linker is attached to the drug via the secondary nitrogen. The resulting conjugates are useful in treating various diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International ApplicationPCT/EP2017/075272, with international filing date of 4 Oct. 2017, whichclaims the benefit of priority to U.S. provisional Application No.62/404,514 filed 5 Oct. 2016, and the contents of each application arehereby incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 3, 2017, isnamed SEQLIST_ST25.txt and 75,169 bytes in size.

FIELD OF THE INVENTION

The subject matter described herein is directed to methods of preparingcertain antibody-drug conjugates (ADCs) wherein the antibody is linkedto the drug through a linker, wherein the drug contains a heteroarylgroup having a secondary nitrogen, and the linker is attached to thedrug via the secondary nitrogen.

BACKGROUND

The major treatment modalities used by oncologists to treat cancer aresurgical resection, radiation therapy, and classical chemotherapeuticdrugs. Unfortunately, surgical resection is not a viable option for manytumors or forms of cancers. Further, radiation therapy andchemotherapeutic drugs do not target only diseased cells, and thereforeit is often the case that damage occurs to off-target healthy cells.

Therapeutics that more specifically target tumor cells are beingdeveloped by taking advantage of tumor-specific expression of antigensor inappropriate overexpression or activation of specific proteinswithin tumor cells. However, tumor cells are prone to mutation and canbecome resistant to drugs that specifically target tumor cells.

Antibody therapy can provide more targeted therapy with less off-targettoxicity. The use of an antibody-drug conjugate (ADC) for the localdelivery of cytotoxic or cytostatic agents can provide delivery of thedrug moiety to tumors, and intracellular accumulation therein. Effortsto design ADCs have focused on the selectivity of monoclonal antibodies(mAbs) as well as drug mechanism of action, drug-linking, anddrug/antibody ratio (loading).

Within the efforts described above, the design of the drug linker is ofimportance, because it impacts both the efficacy and safety of the ADCs.The linker needs to provide sufficient stability during systemiccirculation but allow for the rapid and efficient intracellular releaseof the drug in an active form.

Currently, however, the chemical functionality which may be used informing linkages between a linker and a drug are limited. This limitsboth the linkers and drugs that may be used in ADCs. Therefore, thereexists a need for methodologies which allow for the use of drugs withvaried chemical functionality in the production of ADCs.

BRIEF SUMMARY

In embodiments, the subject matter described herein is directed tomethods of makings ADCs of Formula I.

In embodiments, the subject matter described herein is directed to ADCsof Formula I.

In embodiments, the subject matter described herein is directed tomethods of makings compounds of Formula III.

In embodiments, the subject matter described herein is directed tocompounds of Formula III.

Other embodiments are also described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the reduction of ERα levels in MCF7-neo/HER2cells treated with either CNJ-1 (HER2, circles) or CM-2 (B7H4, squares).

FIG. 2 is a graph showing the reduction of ERα levels in MCF7-neo/HER2cells treated with either CNJ-3 (HER2, circles) or CNJ-4 (B7H4,squares).

FIG. 3 is a set of graphs showing the stability of CNJ-1 measured over24 h in buffer and in whole blood from various species.DAR=antibody-drug conjugate with indicated drug antibody ratio.

FIG. 4 is a set of graphs showing the stability of CNJ-3 measured over24 h in buffer and in whole blood from various species.DAR=antibody-drug conjugate with indicated drug antibody ratio.

FIG. 5 is a graph showing the reduction of ERα levels in MCF7-neo/HER2tumors in mice following single IV treatment with either CNJ-3 (HER2) orCNJ-4 (B7H4). Timepoint=day 4. Error bars=standard error of the mean.

FIG. 6 is a graph showing the reduction of ERα levels in MCF7-neo/HER2tumors in mice following single IV treatment with either CNJ-3 (HER2) orCNJ-4 (B7H4); alternate statistical analysis. Timepoint=day 4. Errorbars=standard error of the mean.

DETAILED DESCRIPTION

Described herein are methods for conjugating antibodies to biologicallyactive molecules through a linker that is covalently bound to asecondary nitrogen contained in the structure of the biologically activemolecule. As set forth below, the methods provide compounds of FormulaeII and III, which are amenable to conjugation with antibodies. Thecompounds of Formulae II and III contain a carbamate formed by asecondary nitrogen on a biologically active molecule and an oxycarbonylon a linker moiety. The compounds of Formulae II and III can then beconjugated with a wide variety of antibodies, engineered antibodies,antibody fragments, etc., to prepare ADCs of Formula I.

The presently disclosed subject matter will now be described more fullyhereinafter. However, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions. Therefore, it is to be understood that the presentlydisclosed subject matter is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims. Inother words, the subject matter described herein covers allalternatives, modifications, and equivalents. In the event that one ormore of the incorporated literature, patents, and similar materialsdiffers from or contradicts this application, including but not limitedto defined terms, term usage, described techniques, or the like, thisapplication controls. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in this field. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety.

I. Definitions

As used herein, the term “alkyl” refers to a straight-chained orbranched hydrocarbon group containing 1 to 12 carbon atoms. Examples ofalkyl groups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, andn-pentyl. Alkyl groups may be optionally substituted with one or moresubstituents.

The term “substituent” refers to an atom or a group of atoms thatreplaces a hydrogen atom on a molecule. The term “substituted” denotesthat a specified molecule bears one or more substituents. Examples ofsubstituents include alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy, alkylthio,sulfonate, amino, alkylamino, acylamino, carbamoyl, alkylcarbamoyl, ornitro.

The term “alkoxy” refers to an —O-alkyl radical. Alkoxy groups may beoptionally substituted with one or more substituents.

The term “haloalkoxy” refers to an —O-alkyl group that is substituted byone or more halo substituents. Examples of haloalkoxy groups includetrifluoromethoxy and 2,2,2-trifluoroethoxy.

The term “arylalkoxy” refers to an —O-alkyl group that is substituted byan aryl substituent. An examples of an arylalkoxy group is O-benzyl.

The term “alkylamino” refers to an amino substituent which is furthersubstituted with one or two alkyl groups.

The term “alkylthio” refers to an —S-alkyl radical. Alkylthio groups maybe optionally substituted with one or more substituents.

The term “acylamino” refers to an amino substituent which is furthersubstituted with a —CO—R group. Examples of acylamino groups includeacetamido and 2-phenylacetamido.

The term “a compound of the formula” or “a compound of formula” or“compounds of the formula” or “compounds of formula” refers to anycompound selected from the genus of compounds as defined by Formula II,III, V, VI, VII, VIII, IX, X, and XI.

The term “benzyl” refers to a hydrocarbon with the formula of C₆H₅CH₂where the point of attachment to the group in question is at the CH₂position. The benzyl may be substituted on the aromatic ring. In oneembodiment, 0, 1, 2, 3, 4, or 5 atoms of the aryl group may besubstituted by a substituent.

As used herein, the term “halogen”, “hal” or “halo” means —F, —Cl, —Bror —I.

The term “cycloalkyl” refers to a hydrocarbon 3-8 membered monocyclic or7-14 membered bicyclic ring system having at least one saturated ring orhaving at least one non-aromatic ring, wherein the non-aromatic ring mayhave some degree of unsaturation. Cycloalkyl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a cycloalkyl group may be substituted by asubstituent. Representative examples of cycloalkyl groups includecyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, cycloheptyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and thelike.

The term “aryl” refers to a hydrocarbon monocyclic, bicyclic ortricyclic aromatic ring system. Aryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, 4, 5 or 6 atoms of each ring of an aryl group may be substituted by asubstituent. Examples of aryl groups include phenyl, naphthyl,anthracenyl, fluorenyl, indenyl, azulenyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-4 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, andthe remainder ring atoms being carbon (with appropriate hydrogen atomsunless otherwise indicated). Heteroaryl groups may have one, two, ormore different tautomeric forms. Heteroaryl groups may be optionallysubstituted with one or more substituents. In one embodiment, 0, 1, 2,3, or 4 atoms of each ring of a heteroaryl group may be substituted by asubstituent. Non-limiting examples of such heteroaryl groups includeimidazolyl, quinolyl, isoquinolyl, indolyl, indazolyl, pyridazyl,pyridyl, pyrrolyl, pyrazolyl, pyrazinyl, quinoxolyl, pyranyl,pyrimidinyl, furyl, thienyl, triazolyl, thiazolyl, carbolinyl,tetrazolyl, benzofuranyl, thiamorpholinyl sulfone, oxazolyl,benzoxazolyl, benzimidazolyl, benzthiazolyl, oxopiperidinyl,oxopyrrolidinyl, oxoazepinyl, azepinyl, isoxazolyl, isothiazolyl,furazanyl, thiadiazyl, oxathiolyl, acridinyl, phenanthridinyl, andbenzocinnolinyl, and the like.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “heterocycloalkyl” refers to a ring or ring system containingat least one heteroatom selected from nitrogen, oxygen, and sulfur,wherein said heteroatom is in a non-aromatic ring. The heterocycloalkylring is optionally fused to or otherwise attached to otherheterocycloalkyl rings and/or non-aromatic hydrocarbon rings and/orphenyl rings. Examples of heterocycloalkyl groups include, for example,piperazinyl, morpholinyl, 1,2,3,4-tetrahydroisoquinolinyl, piperidinyl,tetrahydrofuranyl, pyrrolidinyl, pyridinoyl, and pyrazolidinyl. Theheterocycloalkyl groups may be substituted.

As used herein, unless defined otherwise in a claim, the term“optionally” means that the subsequently described event(s) may or maynot occur, and includes both event(s) that occur and event(s) that donot occur.

As used herein, unless defined otherwise, the phrase “optionallysubstituted”, “substituted” or variations thereof denote an optionalsubstitution, including multiple degrees of substitution, with one ormore substituent group, for example, one, two or three. The phraseshould not be interpreted as duplicative of the substitutions hereindescribed and depicted.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner. As used herein, chiral atoms use the “R” and “S”nomenclature to designate the absolute configuration at said chiralatom.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not minor images of oneanother.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

As used herein, a “leaving group” is the group that is displaced by anucleophile in a conjugation reaction. Leaving groups can be anions orneutral molecules. Anionic leaving groups can be, for example, halidesand sulfonate esters. The group displacing the leaving group can be, forexample, a nucleophile. A “nucleophile” or “nucleophilic group” is achemical species having unshared pair electrons (e.g., any Lewis base),and can be neutral or have a negative charge. A nucleophile donates anelectron pair to form a chemical bond during a chemical reaction.Non-limiting examples of the nucleophilic group include anoxygen-containing group (e.g., hydroxyl, alkoxy, or acyloxy), asulfur-containing group (e.g., mercapto, alkylthio, or sulfonate), anitrogen-containing group (e.g., amino, alkylamino, acylamino, nitro,azido, or isocyanato), and halogen.

The term “protecting group” refers to chemical moieties that are usedduring the preparation of compounds or antibody-drug conjugates forprotection of functionality (e.g., primary or secondary amines,carboxylic acids, or thiols). For a general description of protectinggroups and their use, see T. W. Greene, Protective Groups in OrganicSynthesis, John Wiley & Sons, New York, 1991.

Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (Boc), benzyloxycarbonyl (CBz or CBZ) and9-fluorenylmethyleneoxycarbonyl (Fmoc). Suitable thiol protecting groupsare, for example, unsubstituted or substituted benzyl groups such as abenzyl group, a p-methoxybenzyl group, a 4-methylbenzyl group, a3,4-dimethylbenzyl group, a p-hydroxybenzyl group, a p-acetoxybenzylgroup and a p-nitrobenzyl group, a diphenylmethyl group, a trityl group,a t-butyl group, an acetyl group, a benzoyl group, and so on, with anacid labile protecting group being more preferable. Acid labile thiolprotecting groups can be, for example, trityl, fluorenyl,dimethoxybenzyl, methoxybenzyl, 2,4,6-trimethyl benzyl, xanthenyl,ferrocenyl, methoxymethyl, isobutoxymethyl, and diphenylmethyl. Suitablecarboxylic acid protecting groups can be, for example, branched andunbranched alkyl groups and silyl groups.

In general, the species of protecting group is not critical providedthat it is stable to the conditions of any subsequent reaction(s) onother positions of the compound and can be removed at the appropriatepoint without adversely affecting the remainder of the molecule.

As used herein, the “contacting” refers to reagents in close proximityso that a reaction may occur.

As used herein, “ambient temperature” or “room temperature” refers to atemperature in the range of about 20 to 25° C.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the subject beingtreated therewith.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe subject matter described herein. Exemplary salts include, but arenot limited, to sulfate, citrate, acetate, oxalate, chloride, bromide,iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,lactate, salicylate, acid citrate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucuronate, saccharate, formate, benzoate,glutamate, methanesulfonate “mesylate”, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt, the salt can havemultiple counter ions. Hence, a pharmaceutically acceptable salt canhave one or more charged atoms and/or one or more counter ion.

Other salts, which are not pharmaceutically acceptable, may be useful inthe preparation of compounds of described herein and these should beconsidered to form a further aspect of the subject matter. These salts,such as oxalic or trifluoroacetate, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining the compounds described herein andtheir pharmaceutically acceptable salts.

The components of the ADC can also be described in terms of a “residue,”“moiety” or “group,” which refers to the component being covalentlybound to another component.

The term “covalently bound” or “covalently linked” refers to a chemicalbond formed by sharing of one or more pairs of electrons.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

As used herein, a “reduced antibody” refers to an antibody wherein atleast one cysteine has a free thiol group.

As used herein, the term “acidic conditions” refers to a milieu having apH below 7.0, and specifically conditions that are amenable to cleavageof a leaving group or protecting group, e.g., a pH of less than 1.0,less than 2.0, less than 3.0, less than 4.0, less than 5.0, or less than6.0.

As used herein, a “reducing agent” is a reagent that causes anothersubstance to undergo reduction and that is oxidized in the process. Inthe presence of antibodies, reducing agents can be used to stabilizefree cysteines and to reduce disulfide bonds. Non-limiting examples ofreducing agents are 2-mercaptoethanol, 2-mercaptoethylamine,dithiothreitol, and tris(2-carboxyethyl)phosphine.

As used herein, an “oxidizing agent” is a substance that causes anothersubstance to undergo oxidation and that is reduced in the process. Anon-limiting example of an oxidizing agents is DHAA.

As used herein, a “buffer” is a solution that resists changes in pH whenacid or base is added to it. Buffers typically involve a weak acid orbases together with one of its salts. Non-limiting examples of buffersare Tris, HEPES, PBS (phosphate buffered saline), triethylammoniumacetate buffer, and triethylammonium bicarbonate buffer.

The term “antibody fragment(s)” as used herein comprises a portion of afull length antibody, generally the antigen binding or variable regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies; minibodies (Olafsen et al(2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced bya Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anyof the above which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the subject matter described herein may be made by the hybridomamethod first described by Kohler et al (1975) Nature, 256:495, or may bemade by recombinant DNA methods (see for example: U.S. Pat. Nos.4,816,567; 5,807,715). The monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described in Clacksonet al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA. 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape, etc.) and human constant region sequences.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “intact antibody” as used herein is one comprising a VL and VHdomains, as well as a light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof. The intact antibody may have oneor more “effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

The term “Fc region” as used herein means a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region.

The term includes native sequence Fc regions and variant Fc regions. Inone embodiment, a human IgG heavy chain Fc region extends from Cys226,or from Pro230, to the carboxyl-terminus of the heavy chain. However,the C-terminal lysine (Lys447) of the Fc region may or may not bepresent. Unless otherwise specified herein, numbering of amino acidresidues in the Fc region or constant region is according to the EUnumbering system, also called the EU index, as described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The term “framework” or “FR” as used herein refers to variable domainresidues other than hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4. Accordingly, the HVR and FR sequences generally appear in thefollowing sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Y,where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al (1998) J. Immunol.161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

The term “human consensus framework” as used herein refers to aframework which represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, NIH Publication91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL,the subgroup is subgroup kappa I as in Kabat et al., supra. In oneembodiment, for the VH, the subgroup is subgroup III as in Kabat et al.,supra.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “variable region” or “variable domain” as used herein refers tothe domain of an antibody heavy or light chain that is involved inbinding the antibody to antigen. The variable domains of the heavy chainand light chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). (See,e.g., Kindt et al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co.,page 91 (2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds.

The “epitope 4D5” or “4D5 epitope” or “4D5” is the region in theextracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463)and trastuzumab bind. This epitope is close to the transmembrane domainof HER2, and within domain IV of HER2. To screen for antibodies whichbind to the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 4D5 epitope of HER2 (e.g. any one or more residuesin the region from about residue 550 to about residue 610, inclusive, ofHER2 (SEQ ID NO: 39).

The “epitope 2C4” or “2C4 epitope” is the region in the extracellulardomain of HER2 to which the antibody 2C4 binds. In order to screen forantibodies which bind to the 2C4 epitope, a routine cross-blocking assaysuch as that described in Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprisesresidues from domain II in the extracellular domain of HER2. The 2C4antibody and pertuzumab bind to the extracellular domain of HER2 at thejunction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328(2004)).

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein.

Specific illustrative and exemplary embodiments for measuring bindingaffinity are described in the following. In certain embodiments, anantibody as described herein has dissociation constant (Kd) of ≤1 μM,≤100 nM, ≤10 nM, ≤5 nm, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM,or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g.,from 10⁻⁹ M to 10⁻¹³ M).

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “free cysteine amino acid” as used herein refers to a cysteineamino acid residue which has been engineered into a parent antibody, hasa thiol functional group (—SH), and is not paired as an intramolecularor intermolecular disulfide bridge.

The term “amino acid” as used herein means glycine, alanine, valine,leucine, isoleucine, phenylalanine, proline, serine, threonine,tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan,aspartic acid, glutamic acid, asparagine, glutamine or citrulline.

The term “Linker”, “Linker Unit”, or “link” as used herein means achemical moiety comprising a chain of atoms that covalently attaches adrug to an antibody. In various embodiments, a linker is a divalentradical, specified as L1.

As used herein, the term “plurality” refers to two or more conjugates.Each conjugate can be the same or different from any other conjugate inthe plurality.

Other terms, definitions and abbreviations herein include: Wild-type(“WT”); Cysteine engineered mutant antibody (“thio”); light chain(“LC”); heavy chain (“HC”); 6-maleimidocaproyl (“MC”);maleimidopropanoyl (“MP”); valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe”), p-aminobenzyl (“PAB”), andp-aminobenzyloxycarbonyl (“PABC”); A118C (EU numbering)=A121C(Sequential numbering)=A114C (Kabat numbering) of heavy chain K149C(Kabat numbering) of light chain. Still additional definitions andabbreviations are provided elsewhere herein.

Additional definitions are also provided below.

II. Antibody-Drug-Conjugate (ADCs) and Methods of Preparation

The Antibody-Drug Conjugate (ADC) molecules described herein comprise anantibody conjugated via a linker (L1) to a drug. The general Formula Iof an ADC is:Ab-(L1-D)_(p),  Iwherein, D is a biologically active molecule, e.g. a drug; L1 is alinker, covalently bound to Ab and to D; and p has a value from about 1to about 10, or about 1 to about 9, or about 1 to about 8, or about 1 toabout 7, or about 1 to about 6, or about 1 to about 5, or about 1 toabout 4, or about 1 to about 3. In an embodiment, p is about 2.

The methods described herein are useful for preparing ADCs of Formula Ias well as ADCs wherein more than one, i.e., an integer from 1 to 10,linker-biologically active molecules are conjugated to a single antibodyas in Formula I.

In embodiments, a method for preparing an antibody-drug conjugate ofFormula I:Ab-(L1-D)_(p)  I

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

L1 is a linking moiety;

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl, wherein L1 is covalently bonded to the secondarynitrogen; and

p is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2;

the method comprising:

-   -   i. contacting a compound of Formula II        T-L1-D  II    -   with an antibody, wherein,    -   L1 and D are as described above,    -   T is a leaving group having the structure R⁵—S,        -   wherein R⁵ is an optionally substituted pyridine,    -   wherein an antibody-drug conjugate of Formula I is prepared.        As in any embodiment above, a method wherein

L1 has the structure

wherein,

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, optionally substituted branched or linear C₁-C₅ alkyl, andoptionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring,

-   -   wherein said optionally substituted alkyl or cycloalkyl may be        substituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,        nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy,        alkylthio, sulfonate, amino, alkylamino, acylamino, carbamoyl,        alkylcarbamoyl, or nitro.

The method as in any embodiment above, wherein T-L1-D has the followingformula:

wherein, R⁵ is selected from the group consisting of optionallysubstituted pyridine and nitropyridine.

The method as in any embodiment above, wherein R⁵ is 5-nitropyridine.

The method as in any embodiment above, wherein R¹, R², R³, and R⁴ areindependently selected from the group consisting of H and optionallysubstituted branched or linear C₁-C₅ alkyl.

The method as in any embodiment above, wherein one of R¹, R², R³, and R⁴is optionally substituted branched or linear C₁-C₅ alkyl and the othersare H.

The method as in any embodiment above, wherein the optionallysubstituted branched or linear C₁-C₅ alkyl is methyl.

The method as in any embodiment above, wherein R¹ is methyl, and R², R³,and R⁴ are each H.

The method as in any embodiment above, wherein said antibody-drugconjugate of Formula I has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

The method as in any embodiment above, wherein said antibody-drugconjugate of Formula I has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

The method as in any embodiment above, wherein said Ab is a cysteineengineered antibody or variants thereof.

The method as in any embodiment above, wherein the antibody binds toHER2 or B7-H4.

The method as in any embodiment above, wherein the antibody binds toHER2.

The method as in any embodiment above, wherein said contactingcomprises:

i. contacting said Ab with a suitable reducing agent to prepare areduced Ab,

ii. oxidizing said reduced Ab to prepare Ab′, and

iii. contacting said Ab′ with T-L1-D in the presence of a suitablebuffer.

The method as in any embodiment above, wherein said buffer has a pH ofabout 8.5.

The method as in any embodiment above, wherein said contactingcomprises:

i. contacting said Ab with a molar excess of DTT at ambient temperatureto prepare a reduced Ab,

ii. purifying said reduced Ab,

iii. oxidizing said purified, reduced Ab with DHAA at ambienttemperature to prepare Ab′,

iv. purifying said Ab′,

v. contacting said purified Ab′ with T-L1-D in a buffer solution at a pHof about 8.5 to prepare Ab-L1-D, and

vi. purifying said Ab-L1-D.

The method as in any embodiment above, wherein the carbonyl of L1 iscovalently bound to said secondary nitrogen of the heteroaryl of D,wherein said heteroaryl is selected from the group consisting of:

The method as in any embodiment above, wherein the heteroaryl isselected from the group consisting of:

The method as in any embodiment above, wherein the heteroaryl isselected from the group consisting of:

The method as in any embodiment above, wherein the heteroaryl is

The method as in any embodiment above, wherein D is selected from thegroup consisting of

The method as in any embodiment above, wherein D is

The method as in any embodiment above, wherein D is

The method as in any embodiment above, wherein the antibody-drugconjugate of Formula I is selected from the group consisting of:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

The method as in any embodiment above, wherein said antibody isAnti-HER2 7C2 LC K149C or Anti-B7H4 1D11v1.9 varD LC K149C.]

In embodiments, the subject mater herein is directed to an antibody-drugconjugate of Formula IV:

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, optionally substituted branched or linear C₁-C₅ alkyl, andoptionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring,

-   -   wherein said optionally substituted alkyl or cycloalkyl may be        substituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,        nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy,        alkylthio, sulfonate, amino, alkylamino, acylamino, carbamoyl,        alkylcarbamoyl, or nitro;

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl wherein the carbonyl in Formula IV is covalentlybonded to the secondary nitrogen in D; and

p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about 2.

As in any embodiment above, the antibody-drug conjugate wherein R¹, R²,R³, and R⁴ are independently selected from the group consisting of H andoptionally substituted branched or linear C₁-C₅ alkyl.

As in any embodiment above, the antibody-drug conjugate wherein one ofR¹, R², R³, and R⁴ is optionally substituted branched or linear C₁-C₅alkyl and the others are H.

As in any embodiment above, the antibody-drug conjugate wherein saidoptionally substituted branched or linear C₁-C₅ alkyl is methyl.

As in any embodiment above, the antibody-drug conjugate wherein R¹ ismethyl, and R², R³, and R⁴ are each H.

As in any embodiment above, the antibody-drug conjugate wherein saidantibody-drug conjugate of Formula IV has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

As in any embodiment above, the antibody-drug conjugate, wherein saidantibody-drug conjugate of Formula IV has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

As in any embodiment above, the antibody-drug conjugate wherein said Abis a cysteine engineered antibody or variants thereof.

As in any embodiment above, the antibody-drug conjugate wherein theantibody binds to HER2 or B7-H4.

As in any embodiment above, the antibody-drug conjugate wherein theantibody binds to HER2.

As in any embodiment above, the antibody-drug conjugate wherein thecarbonyl of Formula IV is covalently bound to the secondary nitrogen ofthe heteroaryl of D, wherein the heteroaryl is selected from the groupconsisting of:

As in any embodiment above, the antibody-drug conjugate wherein theheteroaryl is selected from the group consisting of:

As in any embodiment above, the antibody-drug conjugate wherein theheteroaryl is selected from the group consisting of:

As in any embodiment above, the antibody-drug conjugate wherein theheteroaryl is

As in any embodiment above, the antibody-drug conjugate wherein D isselected from the group consisting of

As in any embodiment above, the antibody-drug conjugate wherein D is

As in any embodiment above, the antibody-drug conjugate wherein D is

As in any embodiment above, the antibody-drug conjugate selected fromthe group consisting of:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about2.

As in any embodiment above, the antibody-drug conjugate having thestructure

wherein the antibody is Anti-HER2 7C2 LC K149C and p is 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, and preferably p is about 2.

As in any embodiment above, the antibody-drug conjugate having thestructure

wherein the antibody is Anti-B7H4 1D11v1.9 varD LC K149C and p is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10, and preferably p is about 2.

In embodiments, a method for preparing a compound of Formula III:

wherein,

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, optionally substituted branched or linear C₁-C₅ alkyl, andoptionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring,

-   -   wherein said optionally substituted alkyl or cycloalkyl may be        substituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,        nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy,        alkylthio, sulfonate, amino, alkylamino, acylamino, carbamoyl,        alkylcarbamoyl, or nitro; and

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl wherein the carbonyl in Formula III is covalentlybonded to the secondary nitrogen in D;

R⁵ is selected from the group consisting of optionally substitutedpyridine and nitropyridine;

the method comprising:

i. contacting a compound of Formula V:

wherein LG is a leaving group and PG is a protecting group, with acompound, D, to prepare a compound of Formula VI:

and,

ii. deprotecting the compound of Formula VI under acidic conditions toprepare a compound of Formula III:

The method as in any embodiment above, wherein the protecting group, PG,is a protecting group suitable for thiol protection.

The method as in any embodiment above, wherein the protecting group, PG,is an acid labile protecting group.

The method as in any embodiment above, wherein the protecting group, PG,is selected from the group consisting of trityl, fluorenyl,dimethoxybenzyl, methoxybenzyl, 2, 4, 6-trimethyl benzyl, xanthenyl,ferrocenyl, methoxymethyl, isobutoxymethyl, and diphenylmethyl.

The method as in any embodiment above, wherein the protecting group, PG,is a trityl group.

The method as in any embodiment above, wherein the leaving group, LG, isa group suitable for displacement by a nucleophile.

The method as in any embodiment above, wherein the leaving group, LG, isa halogen.

The method as in any embodiment above, wherein the leaving group, LG, isa chlorine.

The method as in any embodiment above, wherein R⁵ is a nitropyridine.

The method as in any embodiment above, wherein R⁵ is 5-nitropyridine.

In embodiments, a compound of Formula III:

wherein,

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, optionally substituted branched or linear C₁-C₅ alkyl, andoptionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring,

-   -   wherein said optionally substituted alkyl or cycloalkyl may be        substituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,        nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy,        alkylthio, sulfonate, amino, alkylamino, acylamino, carbamoyl,        alkylcarbamoyl, or nitro;

R⁵ is selected from the group consisting of optionally substitutedpyridine and nitropyridine; and

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl wherein the carbonyl in Formula III is covalentlybonded to the secondary nitrogen in D.

As in any embodiment above, the compound wherein R⁵ is 5-nitropyridine.

As in any embodiment above, the compound wherein R¹ is methyl and R²,R³, and R⁴ are each H.

As in any embodiment above, the compound wherein R¹ is methyl and has Rstereochemistry.

As in any embodiment above, wherein the compound of Formula III isselected from the group consisting of

In embodiments, a method for preparing an antibody-drug conjugate ofFormula IV:

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

R¹, R², R³, and R⁴ are independently selected from the group consistingof H, optionally substituted branched or linear C₁-C₅ alkyl, andoptionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring,

-   -   wherein said optionally substituted alkyl or cycloalkyl may be        substituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl,        nitrile, halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy,        alkylthio, sulfonate, amino, alkylamino, acylamino, carbamoyl,        alkylcarbamoyl, or nitro;

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl wherein the carbonyl in Formula IV is covalentlybonded to the secondary nitrogen in D; and

p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably 2;

the method comprising:

i. contacting a compound of Formula V:

with a compound D, wherein LG is a leaving group and PG is a protectinggroup, to prepare a compound of Formula VI:

ii. contacting the compound of Formula VI with a disulfide R⁵—S—S—R⁵under acidic conditions to prepare a compound of Formula III:

wherein R⁵ is an optionally substituted pyridine; and

iii. contacting a compound of Formula III with an antibody to prepare anantibody-drug conjugate of Formula IV.

In embodiments, a method for preparing a compound of Formula VII:

wherein

PG is a protecting group; and

D is a biologically active molecule comprising a secondary nitrogencontaining heteroaryl wherein the ester carbonyl in Formula VII forms acarbamate with the secondary nitrogen in D;

the method comprising:

i. contacting a compound of Formula VIII

with a compound of Formula IX

wherein LG is a leaving group, to prepare a compound of Formula VII.

The method as in any embodiment above, wherein the compound of FormulaVII is

In an embodiment, the antibody-drug conjugate prepared by the method asin any embodiment above.

In an embodiment, a compound of Formula III prepared by the method as inany embodiment above.

The method as in any embodiment above, wherein said heteroaryl isselected from the group consisting of indole, indazole, benzimidazole,benzotriazole, pyrrole, pyrrolopyridine, imidazole, triazole, andtetrazoles.

The method as in any embodiment above, wherein said D is selected fromthe group consisting of cytotoxic agents; growth inhibitory agents;antibiotics; toxins; antitumor or anticancer agents; alkylating agents;alkyl sulfonates; aziridines; ethylenimines and methylamelamines;acetogenins; topoisomerase 1 inhibitors; proteosome inhibitors; EGFRinhibitors; tyrosine kinase inhibitors; serine-threonine kinaseinhibitors; farnesyltransferase inhibitors; anti-hormonal agents;selective estrogen receptor modulators (SERMs); anti-estrogens;aromatase inhibitors; lutenizing hormone-releaseing hormone agonists;sex steroids; estrogens; and androgens/retinoids; estrogen receptordown-regulators (ERDs); anti-androgens; immunosuppressive agents;non-steroidal anti-inflammatory drugs (NSAIDs); anti-inflammatoryagents; cyclooxygenase inhibitors, leukotriene receptor antagonists,purine antagonists; steroids; dihydrofolate reductase inhibitors; andanti-malarial agents.

The method as in any embodiment above, wherein said D is selected fromthe group consisting of amanitin, vinblastine, vincristine, duocarmycinA, duocarmycin SA, CC-1065, adozelesin, U-76074, U-73073, carzelesin(U-80244), KW-2189, diazonamide A, esomeprazole, aripiprazole,valsartan, lansoprazole, rabeprazole, pometrexed, olmesartan, tadalafil,pantoprazole, candosartan, omeprazole, sunitinib, pemetrexed, alectinib,dacarbazine, semaxanib, dacinostat, dovitinib, mebendazole, andpimobendan.

Compounds can be synthesized by synthetic routes that include processesanalogous to those well-known in the chemical arts, particularly inlight of the description contained herein, and those for otherheterocycles described in: Comprehensive Heterocyclic Chemistry II,Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; LiebigsAnnalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database). DTTrefers to dithiothreitol. DHAA refers to dehydroascorbic acid.

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing compounds andnecessary reagents and intermediates are known in the art and include,for example, those described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3^(rd)Ed., John Wiley and Sons(1999); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995) and subsequent editions thereof.

Compounds may be prepared singly or as compound libraries comprising atleast 2, for example 5 to 1,000 compounds, or 10 to 100 compounds.Libraries of compounds of Formula I may be prepared by a combinatorial‘split and mix’ approach or by multiple parallel syntheses using eithersolution phase or solid phase chemistry, by procedures known to thoseskilled in the art. Thus, according to a further aspect, there isprovided a compound library comprising at least 2 compounds, orpharmaceutically acceptable salts thereof.

Methodologies for purifying antibodies are well-known in the art. Anyknown method can be used to purify antibodies, reduced antibodies andthe like as described herein.

The General Procedures and Examples provide exemplary methods forpreparing compounds. Those skilled in the art will appreciate that othersynthetic routes may be used to synthesize the compounds. Althoughspecific starting materials and reagents are depicted and discussed inthe Schemes, General Procedures, and Examples, other starting materialsand reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions. In addition, many of theexemplary compounds prepared by the described methods can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

In general, the present subject matter describes a method for making anADC in a manner such as General Scheme A:

Reaction conditions:

-   -   A: A suitable base in a suitable solvent.    -   B: Reaction conditions providing free cysteine thiol(s) for        reacting with the linker. In one embodiment, step B can        comprise i) contacting Ab with a reducing agent, ii)        purification of the reduced antibody, iii) contacting the        purified antibody with an oxidizing agent to form Ab′, and iv)        purification of Ab′.    -   C: A suitable base in a suitable solvent.

Scheme 1 depicts a synthetic route for preparing a drug disulfide-linkermolecule as described herein.

Scheme 2 depicts a synthetic route for preparing a drug disulfide-linkermolecule as described herein.

Scheme 3 depicts a synthetic route for preparing a drug-protectedpeptide linker molecule as described herein.

Scheme 4 depicts a synthetic route for preparing a drug-peptide linker.

The ADCs described herein are composed of the following components:

1. Antibody (Ab)

As described herein, antibodies, e.g., a monoclonal antibodies (mABs)are used to deliver a drug to target cells, e.g., cells that express thespecific protein that is targeted by the drug.

a. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See. e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

b. Library-Derived Antibodies

Antibodies for use in an ADC may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

c. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody.

Certain chimeric antibodies are described, e.g., in U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). In one example, a chimeric antibody comprises a non-humanvariable region (e.g., a variable region derived from a mouse, rat,hamster, rabbit, or non-human primate, such as a monkey) and a humanconstant region. In a further example, a chimeric antibody is a “classswitched” antibody in which the class or subclass has been changed fromthat of the parent antibody. Chimeric antibodies include antigen-bindingfragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

d. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. The term “multispecific antibody”as used herein refers to an antibody comprising an antigen-bindingdomain that has polyepitopic specificity (i.e., is capable of binding totwo, or more, different epitopes on one molecule or is capable ofbinding to epitopes on two, or more, different molecules).

In some embodiments, multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different antigenbinding sites (such as a bispecific antibody). In some embodiments, thefirst antigen-binding domain and the second antigen-binding domain ofthe multispecific antibody may bind the two epitopes within one and thesame molecule (intramolecular binding). For example, the firstantigen-binding domain and the second antigen-binding domain of themultispecific antibody may bind to two different epitopes on the sameprotein molecule. In certain embodiments, the two different epitopesthat a multispecific antibody binds are epitopes that are not normallybound at the same time by one monospecific antibody, such as e.g. aconventional antibody or one immunoglobulin single variable domain. Insome embodiments, the first antigen-binding domain and the secondantigen-binding domain of the multispecific antibody may bind epitopeslocated within two distinct molecules (intermolecular binding). Forexample, the first antigen-binding domain of the multispecific antibodymay bind to one epitope on one protein molecule, whereas the secondantigen-binding domain of the multispecific antibody may bind to anotherepitope on a different protein molecule, thereby cross-linking the twomolecules.

In some embodiments, the antigen-binding domain of a multispecificantibody (such as a bispecific antibody) comprises two VH/VL units,wherein a first VH/VL unit binds to a first epitope and a second VH/VLunit binds to a second epitope, wherein each VH/VL unit comprises aheavy chain variable domain (VH) and a light chain variable domain (VL).Such multispecific antibodies include, but are not limited to, fulllength antibodies, antibodies having two or more VL and VH domains, andantibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecificdiabodies and triabodies, antibody fragments that have been linkedcovalently or non-covalently). A VH/VL unit that further comprises atleast a portion of a heavy chain variable region and/or at least aportion of a light chain variable region may also be referred to as an“arm” or “hemimer” or “half antibody.” In some embodiments, a hemimercomprises a sufficient portion of a heavy chain variable region to allowintramolecular disulfide bonds to be formed with a second hemimer. Insome embodiments, a hemimer comprises a knob mutation or a holemutation, for example, to allow heterodimerization with a second hemimeror half antibody that comprises a complementary hole mutation or knobmutation. Knob mutations and hole mutations are discussed further below.

In certain embodiments, a multispecific antibody provided herein may bea bispecific antibody. The term “bispecific antibody” as used hereinrefers to a multispecific antibody comprising an antigen-binding domainthat is capable of binding to two different epitopes on one molecule oris capable of binding to epitopes on two different molecules. Abispecific antibody may also be referred to herein as having “dualspecificity” or as being “dual specific.” Exemplary bispecificantibodies may bind both protein and any other antigen. In certainembodiments, one of the binding specificities is for protein and theother is for CD3. See, e.g., U.S. Pat. No. 5,821,337. In certainembodiments, bispecific antibodies may bind to two different epitopes ofthe same protein molecule. In certain embodiments, bispecific antibodiesmay bind to two different epitopes on two different protein molecules.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express protein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al., EMBOJ. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168, WO2009/089004, US2009/0182127, US2011/0287009,Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, andKontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term“knob-into-hole” or “KnH” technology as used herein refers to thetechnology directing the pairing of two polypeptides together in vitroor in vivo by introducing a protuberance (knob) into one polypeptide anda cavity (hole) into the other polypeptide at an interface in which theyinteract. For example, KnHs have been introduced in the Fc:Fc bindinginterfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see.e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, Zhuet al., 1997, Protein Science 6:781-788, and WO2012/106587). In someembodiments, KnHs drive the pairing of two different heavy chainstogether during the manufacture of multispecific antibodies. Forexample, multispecific antibodies having KnH in their Fc regions canfurther comprise single variable domains linked to each Fc region, orfurther comprise different heavy chain variable domains that pair withsimilar or different light chain variable domains. KnH technology can bealso be used to pair two different receptor extracellular domainstogether or any other polypeptide sequences that comprises differenttarget recognition sequences (e.g., including affibodies, peptibodiesand other Fc fusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation.

The term “hole mutation” as used herein refers to a mutation thatintroduces a cavity (hole) into a polypeptide at an interface in whichthe polypeptide interacts with another polypeptide. In some embodiments,the other polypeptide has a knob mutation.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g., by alteringnucleic acid encoding the interface). In some embodiments, nucleic acidencoding the interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The side chain volumes of thevarious amino residues are shown, for example, in Table 1 ofUS2011/0287009. A mutation to introduce a “protuberance” may be referredto as a “knob mutation.”

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiment, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T) and valine (V). In some embodiments, an importresidue is serine, alanine or threonine. In some embodiments, theoriginal residue for the formation of the cavity has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan. Amutation to introduce a “cavity” may be referred to as a “holemutation.”

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG1 constantregion comprises one or more mutations selected from T366S, L368A andY407V (EU numbering). In some embodiments, a hole mutation in an IgG1constant region comprises T366S, L368A and Y407V (EU numbering).

In some embodiments, a knob mutation in an IgG4 constant region is T366W(EU numbering). In some embodiments, a hole mutation in an IgG4 constantregion comprises one or more mutations selected from T366S, L368A, andY407V (EU numbering). In some embodiments, a hole mutation in an IgG4constant region comprises T366S, L368A, and Y407V (EU numbering).

Multispecific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (see, e.g., US2006/0025576A1, and Wu et al. Nature Biotechnology (2007)).). Theantibody or fragment herein also includes a “Dual Acting FAb” or “DAF”comprising an antigen binding site that binds to a target protein aswell as another, different antigen (see, US 2008/0069820, for example).

e. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthin, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

f. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

i. Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Amino acid substitutions. Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

ii. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “THIOMAB™ antibody,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drugs or a Linker L1-drug intermediates, to create an ADC, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A140 (EU numbering) of the heavy chain;L174 (EU numbering) of the heavy chain; Y373 (EU numbering) of the heavychain; K149 (Kabat numbering) of the light chain; A118 (EU numbering) ofthe heavy chain; and S400 (EU numbering) of the heavy chain Fc region.In specific embodiments, the antibodies described herein comprise theHC-A140C (EU numbering) cysteine substitution. In specific embodiments,the antibodies described herein comprise the LC-K149C (Kabat numbering)cysteine substitution. In specific embodiments, the antibodies describedherein comprise the HC-A118C (EU numbering) cysteine substitution.

Cysteine engineered antibodies may be generated as described, e.g., inU.S. Pat. No. 7,521,541.

In certain embodiments, the antibody comprises one of the followingheavy chain cysteine substitutions:

TABLE 2 HC Cysteine Substitutions. Chain EU Mutation Kabat Mutation(HC/LC) Residue Site # Site # HC T 114 110 HC A 140 136 HC L 174 170 HCL 179 175 HC T 187 183 HC T 209 205 HC V 262 258 HC G 371 367 HC Y 373369 HC E 382 378 HC S 424 420 HC N 434 430 HC Q 438 434

In certain embodiments, the antibody comprises one of the followinglight chain cysteine substitutions:

TABLE 3 LC Cysteine Substitutions. Chain EU Mutation Kabat Mutation(HC/LC) Residue Site # Site # LC I 106 106 LC R 108 108 LC R 142 142 LCK 149 149 LC V 205 205

A nonlimiting exemplary hu7C2.v2.2.LA light chain (LC) K149C THIOMAB™antibody has the heavy chain and light chain amino acid sequences of SEQID NOs: 26 and 30, respectively. A nonlimiting exemplary hu7C2.v2.2.LAheavy chain (HC) A118C THIOMAB™ antibody has the heavy chain and lightchain amino acid sequences of SEQ ID NOs: 31 and 25, respectively.

ADCs include cysteine engineered antibodies where one or more aminoacids of a wild-type or parent antibody are replaced with a cysteineamino acid. Any form of antibody may be so engineered, i.e. mutated. Forexample, a parent Fab antibody fragment may be engineered to form acysteine engineered Fab, referred to herein as “ThioFab.” Similarly, aparent monoclonal antibody may be engineered to form a “ThioMab.” Itshould be noted that a single site mutation yields a single engineeredcysteine residue in a ThioFab, while a single site mutation yields twoengineered cysteine residues in a ThioMab, due to the dimeric nature ofthe IgG antibody. Mutants with replaced (“engineered”) cysteine (Cys)residues are evaluated for the reactivity of the newly introduced,engineered cysteine thiol groups. The thiol reactivity value is arelative, numerical term in the range of 0 to 1.0 and can be measuredfor any cysteine engineered antibody. Thiol reactivity values ofcysteine engineered antibodies for use in an ADC are in the ranges of0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0.

To prepare a cysteine engineered antibody by mutagenesis, DNA encodingan amino acid sequence variant of the starting polypeptide is preparedby a variety of methods known in the art. These methods include, but arenot limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may be constructed also byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. Mutagenic primers encode the cysteine codonreplacement(s). Standard mutagenesis techniques can be employed togenerate DNA encoding such mutant cysteine engineered antibodies.General guidance can be found in Sambrook et al Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel et al Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485;WO2009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191;Junutula et al (2008) Jour of Immun. Methods 332:41-52). The engineeredcysteine thiols may react with linker reagents or the Linker-Drugintermediates described herein, which have thiol-reactive, electrophilicgroups such as maleimide or alpha-halo amides to form an ADC withcysteine engineered antibodies (ThioMabs) and the drug residue. Thelocation of the drug moiety can thus be designed, controlled, and known.Drug/antibody ratio (“PAR”) can be controlled since the engineeredcysteine thiol groups typically react with thiol-reactive linkerreagents or linker-drug intermediates in high yield. Engineering anantibody to introduce a cysteine amino acid by substitution at a singlesite on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A PAR of about 2 can be achieved and nearhomogeneity of the conjugation product.

Cysteine engineered antibodies preferably retain the antigen bindingcapability of their wild type, parent antibody counterparts. Thus,cysteine engineered antibodies are capable of binding, preferablyspecifically, to antigens. Such antigens include, for example,tumor-associated antigens (TAA), cell surface receptor proteins andother cell surface molecules, transmembrane proteins, signalingproteins, cell survival regulatory factors, cell proliferationregulatory factors, molecules associated with (for e.g., known orsuspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein). An antigen to which acysteine engineered antibody is capable of binding may be a member of asubset of one of the above-mentioned categories, wherein the othersubset(s) of said category comprise other molecules/antigens that have adistinct characteristic (with respect to the antigen of interest).

Cysteine engineered antibodies are prepared for conjugation with linkeror linker-drug intermediates by reduction and reoxidation of intrachaindisulfide groups.

iii. Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody may be made in order to create antibodyvariants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

iv. Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the subject matter described herein is directedto an antibody variant that possesses some but not all effectorfunctions, which make it a desirable candidate for applications in whichthe half-life of the antibody in vivo is important yet certain effectorfunctions (such as complement and ADCC) are unnecessary or deleterious.In vitro and/or in vivo cytotoxicity assays can be conducted to confirmthe reduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)).

Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

In some embodiments, one or more amino acid modifications may beintroduced into the Fc portion of the antibody provided herein in orderto increase IgG binding to the neonatal Fc receptor. In certainembodiments, the antibody comprises the following three mutationsaccording to EU numbering: M252Y, S254T, and T256E (the “YTE mutation”)(U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal ofBiological Chemistry 281(33):23514-23524 (2006). In certain embodiments,the YTE mutation does not affect the ability of the antibody to bind toits cognate antigen. In certain embodiments, the YTE mutation increasesthe antibody's serum half-life compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by 3-fold compared to the native (i.e.,non-YTE mutant) antibody. In some embodiments, the YTE mutationincreases the serum half-life of the antibody by 2-fold compared to thenative (i.e., non-YTE mutant) antibody. In some embodiments, the YTEmutation increases the serum half-life of the antibody by 4-foldcompared to the native (i.e., non-YTE mutant) antibody. In someembodiments, the YTE mutation increases the serum half-life of theantibody by at least 5-fold compared to the native (i.e., non-YTEmutant) antibody. In some embodiments, the YTE mutation increases theserum half-life of the antibody by at least 10-fold compared to thenative (i.e., non-YTE mutant) antibody. See, e.g., U.S. Pat. No.8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant provides a means to modulateantibody-dependent cell-mediated cytotoxicity (ADCC) activity of theantibody. In certain embodiments, the YTEO mutant provides a means tomodulate ADCC activity of a humanized IgG antibody directed against ahuman antigen. See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acquaet al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

In certain embodiments, the YTE mutant allows the simultaneousmodulation of serum half-life, tissue distribution, and antibodyactivity (e.g., the ADCC activity of an IgG antibody). See, e.g., U.S.Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of BiologicalChemistry 281(33):23514-23524 (2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 according to EU numbering (U.S. Pat. No. 6,737,056).Such Fc mutants include Fc mutants with substitutions at two or more ofamino acid positions 265, 269, 270, 297 and 327 according to EUnumbering, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine according to EU numbering (i.e., D265Aand N297A according to EU numbering) (U.S. Pat. No. 7,332,581). Incertain embodiments the Fc mutant comprises the following two amino acidsubstitutions: D265A and N297A. In certain embodiments the Fc mutantconsists of the following two amino acid substitutions: D265A and N297A.

In certain embodiments, the proline at position 329 (EU numbering)(P329) of a wild-type human Fc region is substituted with glycine orarginine or an amino acid residue large enough to destroy the prolinesandwich within the Fc/Fcγ receptor interface, that is formed betweenthe P329 of the Fc and tryptophane residues W87 and W110 of FcgRIII(Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In a furtherembodiment, at least one further amino acid substitution in the Fcvariant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S andstill in another embodiment said at least one further amino acidsubstitution is L234A and L235A of the human IgG1 Fc region or S228P andL235E of the human IgG4 Fc region, all according to EU numbering (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In certain embodiments, a polypeptide comprises the Fc variant of awild-type human IgG Fc region wherein the polypeptide has P329 of thehuman IgG Fc region substituted with glycine and wherein the Fc variantcomprises at least two further amino acid substitutions at L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EUnumbering (U.S. Pat. No. 8,969,526 which is incorporated by reference inits entirety). In certain embodiments, the polypeptide comprising theP329G, L234A and L235A (EU numbering) substitutions exhibit a reducedaffinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCCto at least 20% of the ADCC induced by the polypeptide comprising thewild-type human IgG Fc region, and/or for down-modulation of ADCP (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In a specific embodiment the polypeptide comprising an Fc variant of awild-type human Fc polypeptide comprises a triple mutation: an aminoacid substitution at position Pro329, a L234A and a L235A mutationaccording to EU numbering (P329/LALA) (U.S. Pat. No. 8,969,526 which isincorporated by reference in its entirety). In specific embodiments, thepolypeptide comprises the following amino acid substitutions: P329G,L234A, and L235A according to EU numbering.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826)according to EU numbering. See also Duncan & Winter, Nature 322:738-40(1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerningother examples of Fc region variants.

g. Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

h. Tumor-Associated Antigens

Antibodies, including but not limited to cysteine engineered antibodies,which may be useful in the ADCs described herein in the treatment ofcancer include, but are not limited to, antibodies against cell surfacereceptors and tumor-associated antigens (TAA). Certain tumor-associatedantigens are known in the art, and can be prepared for use in generatingantibodies using methods and information which are well known in theart. In attempts to discover effective cellular targets for cancerdiagnosis and therapy, researchers have sought to identify transmembraneor otherwise tumor-associated polypeptides that are specificallyexpressed on the surface of one or more particular type(s) of cancercell as compared to on one or more normal non-cancerous cell(s). Often,such tumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability to morespecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, those listed below. For convenience, information relating to theseantigens, all of which are known in the art, is listed below andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA listed below areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,and/or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

i. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an antibody described herein is provided.Such nucleic acid may encode an amino acid sequence comprising the VLand/or an amino acid sequence comprising the VH of the antibody (e.g.,the light and/or heavy chains of the antibody). In a further embodiment,one or more vectors (e.g., expression vectors) comprising such nucleicacid are provided. In a further embodiment, a host cell comprising suchnucleic acid is provided. In one such embodiment, a host cell comprises(e.g., has been transformed with): (1) a vector comprising a nucleicacid that encodes an amino acid sequence comprising the VL of theantibody and an amino acid sequence comprising the VH of the antibody,or (2) a first vector comprising a nucleic acid that encodes an aminoacid sequence comprising the VL of the antibody and a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VH of the antibody. In one embodiment, the host cell is eukaryotic,e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,Sp20 cell). In one embodiment, a method of making an antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an antibody, nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977);baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells;and FS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Referring now to antibody affinity, in embodiments, the antibody bindsto one or more tumor-associated antigens or cell-surface receptorsselected from (1)-(53):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203)

ten Dijke, P., et al Science 264 (5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004063362 (Claim 2); WO2003042661 (Claim 12);US2003134790-A1 (Page 38-39); WO2002102235 (Claim 13; Page 296);WO2003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530);WO2003029421 (Claim 6); WO2003024392 (Claim 2; FIG. 112); WO200298358(Claim 1; Page 183); WO200254940 (Page 100-101); WO200259377 (Page349-350); WO200230268 (Claim 27; Page 376); WO200148204 (Example; FIG.4);NP_001194 bone morphogenetic protein receptor, type IB/pid=NP_001194.1Cross-references: MIM:603248; NP_001194.1; AY065994(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem.Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291(1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33;Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (Claim 12; Page 150);NP_003477 solute carrier family 7 (cationic amino acid transporter,y+system), member 5/pid=NP_003477.3—Homo sapiensCross-references: MIM:600182; NP_003477.3; NM_015923; NM_003486_1(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_012449)Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc.Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004065577 (Claim 6);WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (Claim 2);WO2003042661 (Claim 12); US2003157089 (Example 5); US2003185830 (Example5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619);WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG.2A);NP_036581 six transmembrane epithelial antigen of the prostateCross-references: MIM:604415; NP_036581.1; NM_012449_1(4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J. Biol. Chem.276 (29):27371-27375 (2001)); WO2004045553 (Claim 14); WO200292836(Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121); US2003124140(Example 16); U.S. Pat. No. 798,959. Cross-references: GI:34501467;AAK74120.3; AF361486_1(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_005823) Yamaguchi, N., et al Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14);(WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_005814.2; NM_005823_1(6) Napi2b (Napi3b, NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34(sodium phosphate), member 2, type II sodium-dependent phosphatetransporter 3b, Genbank accession no. NM_006424)J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (Claim 2); EP1394274 (Example 11);WO2002102235 (Claim 13; Page 326); EP875569 (Claim 1; Page 17-19);WO200157188 (Claim 20; Page 329); WO2004032842 (Example IV); WO200175177(Claim 24; Page 139-140); Cross-references: MIM:604217; NP 006415.1;NM_006424_1(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878)Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1);WO2003003984 (Claim 1); WO200206339 (Claim 1; Page 50); WO200188133(Claim 1; Page 41-43, 48-58); WO2003054152 (Claim 20); WO2003101400(Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737;(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002)Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056(Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5);EP1347046 (Claim 1); WO2003025148 (Claim 20);Cross-references: GI:37182378; AAQ88991.1; AY358628_1(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938 (Example2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475(Claim 1); WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494(FIG. 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page124-125); EP522868 (Claim 8; FIG. 2); WO200177172 (Claim 1; Page297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.5,773,223 (Claim 1a; Col 31-34); WO2004001004;(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_017763);WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim12); WO2003083074 (Claim 14; Page 61); WO2003018621 (Claim 1);WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894: NP_060233.2; NM_017763_1(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138)Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397(Claim 1; FIG. 1); WO200272596 (Claim 13; Page 54-55); WO200172962(Claim 1; FIG. 4B); WO2003104270 (Claim 11); WO2003104270 (Claim 16);US2004005598 (Claim 22); WO2003042661 (Claim 12); US2003060612 (Claim12; FIG. 10); WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12;FIG. 10); Cross-references: GI:22655488; AAN04080.1; AF455138_1(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_017636)Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003143557 (Claim 4); WO200040614 (Claim 14; Page 100-103);WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12); WO200230268(Claim 27; Page 391); US2003219806 (Claim 4); WO200162794 (Claim 14;FIG. 1A-D);Cross-references: MIM:606936; NP_060106.2; NM_017636_1(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_003203 or NM_003212)Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2);Cross-references: MIM: 187395; NP 003203.1; NM_003212_1(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004)Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad.Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595(Claim 1);Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.(15) CD79b (CD79B, CD7913, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_000626 or 11038674)Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625);WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1,page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573(claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003048202 (claim 1,pages 306 and 309); WO 99/558658, U.S. Pat. No. 6,534,482 (claim 13,FIG. 17A/B); WO200055351 (claim 11, pages 1145-1146);Cross-references: MIM:147245; NP_000617.1; NM_000626_1(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764,AY358130)Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95(2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98(17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem. Biophys. Res.Commun. 280 (3):768-775; WO2004016225 (Claim 2); WO2003077836;WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803 (Claim 12);WO2003089624 (Claim 25);Cross-references: MIM:606509; NP_110391.2; NM_030764_1(17) HER2 (ErbB2, Genbank accession no. M11730)Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., etal Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci.U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165,869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999;Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993)Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I);WO2004009622; WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim1); US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4);Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.(18) NCA (CEACAM6, Genbank accession no. M18728);Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc.Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393(Claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim12); WO200278524 (Example 2); WO200286443 (Claim 27; Page 427),WO200260317 (Claim 2);Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;(19) MDP (DPEP1, Genbank accession no. BC017023)Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003016475(Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (FIG. 6-8);WO9946284 (FIG. 9);Cross-references: MIM: 179780; AAH17023.1; BC017023_1(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et alNature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001;Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al GenomeRes. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci.U.S.A. 99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373(Claim 11); US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG.52); US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52);US2003119129 (Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1;FIG. 52); US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634(Claim 1);(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_004442)Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10(5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev.Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12); WO200053216 (Claim1; Page 41); WO2004065576 (Claim 1); WO2004020583 (Claim 9);WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42);Cross-references: MIM:600997; NP_004433.2; NM_004442_1(23) ASLG659 (B7h, Genbank accession no. AX092328)US20040101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3);US2003165504 (Claim 1); US2003124140 (Example 2); US2003065143 (FIG.60); WO2002102235 (Claim 13; Page 299); US2003091580 (Example 2);WO200210187 (Claim 6; FIG. 10); WO200194641 (Claim 12; FIG. 7b);WO200202624 (Claim 13; FIG. 1A-IB); US2002034749 (Claim 54; Page 45-46);WO200206317 (Example 2; Page 320-321, Claim 34; Page 321-322);WO200271928 (Page 468-469); WO200202587 (Example 1; FIG. 1); WO200140269(Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207);WO2004053079 (Claim 12); WO2003004989 (Claim 1); WO200271928 (Page233-234, 452-453); WO 0116318;(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436)Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998;Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res.Commun. (2000) 275(3):783-788; WO2004022709; EP1394274 (Example 11);US2004018553 (Claim 17); WO2003008537 (Claim 1); WO200281646 (Claim 1;Page 164); WO2003003906 (Claim 10; Page 288); WO200140309 (Example 1;FIG. 17); US2001055751 (Example 1; FIG. 1b); WO200032752 (Claim 18; FIG.1); WO9851805 (Claim 17; Page 97); WO9851824 (Claim 10; Page 94);WO9840403 (Claim 2; FIG. 1B);Accession: 043653; EMBL; AF043498; AAC39607.1.(25) GEDA (Genbank accession No. AY260763);AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1—HomosapiensSpecies: Homo sapiens (human); WO2003054152 (Claim 20); WO2003000842(Claim 1); WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45);Cross-references: GI:30102449; AAP14954.1; AY260763_1(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—HomosapiensThompson, J. S., et al Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1;NM_052945_1; AF132600 (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM,Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilsonet al (1991) J. Exp. Med. 173:137-146; WO2003072036 (Claim 1, FIG. 1);Cross-references: MIM:107266; NP 001762.1; NM_001771_1(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_001774.10)WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S.Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531;Mueller et al (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al(1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al (1988) EMBO J. 7(11):3457-3464; (29) CXCR5 (Burkitt'slymphoma receptor 1, a G protein-coupled receptor that is activated bythe CXCL13 chemokine, functions in lymphocyte migration and humoraldefense, plays a role in HIV-2 infection and perhaps development ofAIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7[P] Gene Chromosome: 11q23.3, Genbank accession No. NP_001707.1)WO2004040000; WO2004015426; US2003105292 (Example 2); U.S. Pat. No.6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188 (Claim 20, page269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153,Example 2, pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No.5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497(claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol. 22:2795-2799;Barella et al (1995) Biochem. J. 309:773-779;(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111.1)Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119;(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_002552.2)Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035(claim 10); Touchman et al (2000) Genome Res. 10:165-173; WO200222660(claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277(page 82);(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1 . . . 359; 359 aa), pI: 8.66, MW: 40225TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1)WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877;Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_005573.1)US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996)Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages24-26);(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_443170.1)WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al(2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim8); EP1347046 (claim 1); WO2003089624 (claim 7);(35) FCRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2, a putative immunoreceptor with possible roles in B celldevelopment and lymphomagenesis; deregulation of the gene bytranslocation occurs in some B cell malignancies); 977 aa, pI: 6.88 MW:106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No.Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453,AK090423, AK090475, AL834187, AY358085; Mouse:AK089756, AY158090,AY506558; NP 112571.1WO2003024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem. Biophys.Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG.18B-1-18B-2);(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;Genbank accession No. AF179274; AY358907, CAF85723, CQ782436WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8);WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944(pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706);US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152;Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang etal (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int JCancer. October 15; 94(2): 178-84;(37) PMELI7 (silver homolog; SILV; D12S53E; PMELI7; SI; SIL); ME20;gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R. P.et al (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736;Kummer, M. P. et al (2009) J. Biol. Chem. 284 (4), 2296-2306;(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains 1; Tomoregulin-1); H7365; C9orf2; C90ORF2;U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al (2003) Oncogene 22 (18):2723-2727;(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962;NM_145793 NM_005264; Kim, M. H. et al (2009) Mol. Cell. Biol. 29 (8),2264-2277; Treanor, J. J. et al (1996) Nature 382 (6586):80-83;(40) Ly6E (lymphocyte antigen 6 complex, locus E, Ly67, RIG-E, SCA-2,TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A. G. et al (2003)Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et al (2002) Mol. Cell.Biol. 22 (3):946-952; WO 2013/17705;(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_001007539.1;NM_001007538.1; Furushima, K. et al (2007) Dev. Biol. 306 (2), 480-492;Clark, H. F. et al (2003) Genome Res. 13 (10):2265-2270;(42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80(1):113-123; Ribas, G. et al (1999) J. Immunol. 163 (1):278-287;(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al (2009) Am. J.Epidemiol. 170 (5):537-545; Yamamoto, Y. et al (2003) Hepatology 37(3):528-533;(44) RET (ret proto-oncogene; MEN2A; HSCRI; MEN2B; MTC1; PTC; CDHF12;Hs. 168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4; Tsukamoto, H. etal (2009) Cancer Sci. 100 (10):1895-1901; Narita, N. et al (2009)Oncogene 28 (34):3058-3068;(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al (2007) CancerRes. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et al (2003) Int. J.Cancer 103 (6):768-774;(46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1;NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O'Dowd, B. F. et al (1996) FEBS Lett. 394 (3):325-329;(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);NP_115940.2; NM_032551.4; Navenot, J. M. et al (2009) Mol. Pharmacol. 75(6):1300-1306; Hata, K. et al (2009) Anticancer Res. 29 (2):617-623;(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);NP_859069.2; NM_181718.3; Gerhard, D. S. et al (2004) Genome Res. 14(10B):2121-2127;(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1;NM_000372.4; Bishop, D. T. et al (2009) Nat. Genet. 41 (8):920-925; Nan,H. et al (2009) Int. J. Cancer 125 (4):909-917;(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);NP_001103373.1; NM_001109903.1; Clark, H. F. et al (2003) Genome Res. 13(10):2265-2270; Scherer, S. E. et al (2006) Nature 440 (7082):346-351(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A. et al (2003)Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et al(2002) FEBS Lett. 520 (1-3):97-101.(52) CD33, a member of the sialic acid binding, immunoglobulin-likelectin family, is a 67-kDa glycosylated transmembrane protein. CD33 isexpressed on most myeloid and monocytic leukemia cells in addition tocommitted myelomonocytic and erythroid progenitor cells. It is not seenon the earliest pluripotent stem cells, mature granulocytes, lymphoidcells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin.Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33contains two tyrosine residues on its cytoplasmic tail, each of which isfollowed by hydrophobic residues similar to the immunoreceptortyrosine-based inhibitory motif (ITIM) seen in many inhibitoryreceptors.(53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of the C-typelectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of thisfamily share a common protein fold and have diverse functions, such ascell adhesion, cell-cell signalling, glycoprotein turnover, and roles ininflammation and immune response. The protein encoded by this gene is anegative regulator of granulocyte and monocyte function. Severalalternatively spliced transcript variants of this gene have beendescribed, but the full-length nature of some of these variants has notbeen determined. This gene is closely linked to other CTL/CTLDsuperfamily members in the natural killer gene complex region onchromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90; van Rhenen A, et al., (2007) Blood 110 (7):2659-66; Chen CH, et al. (2006) Blood 107 (4):1459-67; Marshall A S, et al. (2006) Eur.J. Immunol. 36 (8):2159-69; Bakker A B, et al (2005) Cancer Res. 64(22):8443-50; Marshall A S, et al (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembranereceptor comprising a single C-type lectin-like domain (which is notpredicted to bind either calcium or sugar), a stalk region, atransmembrane domain and a short cytoplasmic tail containing an ITIMmotif.

As described herein, an ADC comprises an antibody, e.g., an antibodyselected from:

Anti-Ly6E Antibodies

In certain embodiments, an ADC can comprise anti-Ly6E antibodies.Lymphocyte antigen 6 complex, locus E (Ly6E), also known as retinoicacid induced gene E (RIG-E) and stem cell antigen 2 (SCA-2). It is a GPIlinked, 131 amino acid length, ˜8.4 kDa protein of unknown function withno known binding partners. It was initially identified as a transcriptexpressed in immature thymocyte, thymic medullary epithelial cells inmice (Mao, et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:5910-5914). Insome embodiments, the subject matter described herein provides an ADCcomprising an anti-Ly6E antibody described in PCT Publication No. WO2013/177055.

In some embodiments, the subject matter described herein provides an ADCcomprising an anti-Ly6E antibody comprising at least one, two, three,four, five, or six HVRs selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 14; (d) HVR-L1 comprising the amino acid sequence of SEQID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In one aspect, the subject matter described herein provides an ADCcomprising an antibody that comprises at least one, at least two, or allthree VH HVR sequences selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 13; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 14. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:12; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

In another aspect, the subject matter described herein provides an ADCcomprising an antibody that comprises at least one, at least two, or allthree VL HVR sequences selected from (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 11. In one embodiment, the antibody comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 12, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 13, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 14; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 10, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 11.

In another aspect, the subject matter described herein provides an ADCcomprising an antibody that comprises (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 12; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 14; (d) HVR-L1 comprising the amino acid sequence of SEQID NO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:10; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In any of the above embodiments, an anti-Ly6E antibody of an ADC ishumanized. In one embodiment, an anti-Ly6E antibody comprises HVRs as inany of the above embodiments, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework.

In another aspect, an anti-Ly6E antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 8. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO:8 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-Ly6E antibodycomprising that sequence retains the ability to bind to Ly6E. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 8. In certain embodiments, a totalof 1 to 5 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 8. In certain embodiments, substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-Ly6E antibody comprises the VH sequence of SEQ IDNO: 8, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12,(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

In another aspect, an anti-Ly6E antibody of an ADC is provided, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 7. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:7contains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-Ly6Eantibody comprising that sequence retains the ability to bind to Ly6E.In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 7. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 7. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-Ly6E antibody comprisesthe VL sequence of SEQ ID NO: 7, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 9; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 10; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 11.

In another aspect, an ADC comprising an anti-Ly6E antibody is provided,wherein the antibody comprises a VH as in any of the embodimentsprovided above, and a VL as in any of the embodiments provided above.

In one embodiment, an ADC is provided, wherein the antibody comprisesthe VH and VL sequences in SEQ ID NO: 8 and SEQ ID NO: 7, respectively,including post-translational modifications of those sequences.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-Ly6E antibody provided herein. Forexample, in certain embodiments, an ADC is provided comprising anantibody that binds to the same epitope as an anti-Ly6E antibodycomprising a VH sequence of SEQ ID NO: 8 and a VL sequence of SEQ ID NO:7, respectively.

In a further aspect, an anti-Ly6E antibody of an ADC according to any ofthe above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-Ly6E antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein. In some embodiments, an ADCcomprises an anti-Ly6E antibody comprising a heavy chain and a lightchain comprising the amino acid sequences of SEQ ID NO: 16 and 15,respectively.

TABLE 4 Ly6E Antibody Sequences. SEQ ID NO Description Sequence  7anti-Ly6E DIQMTQSPSS LSASVGDRVT ITCSASQGIS NYLNWYQQKP antibodyGKTVKLLIYY TSNLHSGVPS RFSGSGSGTD YTLTISSLQP hu9B12 v12EDFATYYCQQ YSELPWTFGQ GTKVEIK light chain variable region  8 anti-Ly6EEVQLVESGPA LVKPTQTLTL TCTVSGFSLT GYSVNWIRQPGKAL antibodyEWLGMIWGDG STDYNSALKS RLTISKDTSK NQVVLTMTNM hu9B12 v12DPVDTATYYC ARDYYFNYAS WFAYWGQGTL VTVSS heavy chain variable region  9anti-Ly6E SASQGISNYLN antibody hu9B12 v12 HVR-L1 10 anti-Ly6E YTSNLHSantibody hu9B12 v12 HVR-L2 11 anti-Ly6E QQYSELPWT antibody hu9B12 v12HVR-L3 12 anti-Ly6E GFSLTGYSVN antibody hu9B12 V12 HVR-H1 13 anti-Ly6EMIWGDGSTDY NSALKS antibody hu9B12 v12 HVR-H2 14 anti-Ly6E DYYVNYASWEAYantibody hu9B12 v12 HVR-H3 15 anti-Ly6EDIQMTQSPSS LSASVGDRVT ITCSASQGIS NYLNWYQQKP antibodyGKTVKLLIYY TSNLHSGVPS RFSGSGSGTD YTLTISSLQP hu9B12 v12EDFATYYCQQ YSELPWTFGQ GTKVEIK RTVAAPSVFIF K149C kappaPPSDEQLKSG TASVVCLLNN FYPREAKVQW CVDNALQSGN light chainSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC 16anti-Ly6E EVQLVESGPA LVKPTQTLTL TCTVSGFSLT GYSVNWIRQP antibodyPGKALEWLGM IWGDGSTDYN SALKSRLTIS KDTSKNQVVL hu9B12 v12TMTNMDPVDT ATYYCARDYY FNYASWFAYW GQGTLVTVSS IgG1 heavyASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS chainWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGKAnti-HER2 Antibodies

In certain embodiments, ADCs comprise anti-HER2 antibodies. In oneembodiment, an anti-HER2 antibody of an ADC comprises a humanizedanti-HER2 antibody, e.g., huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8, asdescribed in Table 3 of U.S. Pat. No. 5,821,337, which is specificallyincorporated by reference herein. Those antibodies contain humanframework regions with the complementarity-determining regions of amurine antibody (4D5) that binds to HER2. The humanized antibodyhuMAb4D5-8 is also referred to as trastuzumab, commercially availableunder the tradename HERCEPTIN®. In another embodiment, an anti-HER2antibody of an ADC comprises a humanized anti-HER2 antibody, e.g.,humanized 2C4, as described in U.S. Pat. No. 7,862,817. An exemplaryhumanized 2C4 antibody is pertuzumab, commercially available under thetradename PERJETA®.

In another embodiment, an anti-HER2 antibody of an ADC comprises ahumanized 7C2 anti-HER2 antibody. A humanized 7C2 antibody is ananti-HER2 antibody.

In some embodiments, described herein are ADCs comprising an anti-HER2antibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23,27, or 28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24 or 29; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:19: (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In someembodiments, described herein are ADCs comprising an anti-HER2 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 21.

In one aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VH HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23,27, or 28; and (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 24 or 29. In one aspect, described herein are ADCs comprising anantibody that comprises at least one, at least two, or all three VH HVRsequences selected from (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 23; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:24. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 68, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 23, 27, or 28; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or 29. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 23; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 24.

In another aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 19; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. Inone embodiment, the antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 19: (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 22, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 23, 27, or 28, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 24 or 29; and (b) a VL domain comprising atleast one, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 20, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 21. In another aspect,an ADC comprises an antibody comprising (a) a VH domain comprising atleast one, at least two, or all three VH HVR sequences selected from (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 23, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO: 24; and (b) aVL domain comprising at least one, at least two, or all three VL HVRsequences selected from (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 19, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 20, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:21.

In another aspect, described herein are ADCs comprising an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23, 27,or 28; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 or29; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 19; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 20; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In anotheraspect, described herein are ADCs comprising an antibody that comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 19; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 20; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 21.

In any of the above embodiments, an anti-HER2 antibody of an ADC ishumanized. In one embodiment, an anti-HER2 antibody of an ADC comprisesHVRs as in any of the above embodiments, and further comprises a humanacceptor framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In another aspect, an anti-HER2 antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98°/%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO: 18 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-HER2 antibodycomprising that sequence retains the ability to bind to HER2. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 18. In certain embodiments, atotal of 1 to 5 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 18. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-HER2 antibody comprises the VH sequence ofSEQ ID NO: 18, including post-translational modifications of thatsequence. In a particular embodiment, the VH comprises one, two or threeHVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 22, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:23, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In another aspect, an anti-HER2 antibody of an ADC is provided, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 17. In certainembodiments, a VL sequence having at least 900/%, 91⁰%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ IDNO: 17 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-HER2 antibody comprising that sequence retains the ability to bindto HER2. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 17. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 17. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-HER2 antibody comprisesthe VL sequence of SEQ ID NO: 17, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 19; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 20; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 21.

In another aspect, an ADC comprising an anti-HER2 antibody is provided,wherein the antibody comprises a VH as in any of the embodimentsprovided above, and a VL as in any of the embodiments provided above.

In one embodiment, an ADC comprising an antibody is provided, whereinthe antibody comprises the VH and VL sequences in SEQ ID NO: 18 and SEQID NO: 17, respectively, including post-translational modifications ofthose sequences.

In one embodiment, an ADC comprising an antibody is provided, whereinthe antibody comprises the humanized 7C2.v2.2.LA (hu7C2) K149C kappalight chain sequence of SEQ ID NO: 30.

In one embodiment, an ADC comprising an antibody is provided, whereinthe antibody comprises the Hu7C2 A118C IgG1 heavy chain sequence of SEQID NO: 31.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-HER2 antibody provided herein. Forexample, in certain embodiments, an ADC is provided, comprising anantibody that binds to the same epitope as an anti-HER2 antibodycomprising a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ IDNO: 17, respectively.

In a further aspect, an anti-HER2 antibody of an ADC according to any ofthe above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-HER2 antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, an ADC comprises an antibody that is asubstantially full length antibody, e.g., an IgG1 antibody, IgG2aantibody or other antibody class or isotype as defined herein.

TABLE 5 Humanized 7C2 anti-HER2 antibody sequences. SEQ. ID NO.Description Sequence 17 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY 7C2.v2.2.LAQQKPGQPPKL LIKYASILES GVPDRFSGSG SGTDFTLTIS (″hu7C2″)SLQAEDVAVY YCQHSWEIPP WTFGQGTKVE IK light chain variable region 18Hu7C2 heavy EVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA chain variablePGQGLEWIGM IHPLDAEIRA NQKFRDRVTI TVDTSTSTAY regionLELSSLRSED TAVYYCARGT YDGGFEYWGQ GTLVTVSS 19 Hu7C2 HVR- RASQSVSGSRFTYMHL1 20 Hu7C2 HVR- YASILES L2 21 Hu7C2 HVR- QHSWEIPPWT L3 22 Hu7C2 HVR-GYWMN H1 23 Hu7C2 HVR- MIHPLDAEIRANQKFRD H2 (Hu7C2. v2.1.S53L, S55A HVR-H2) 24 Hu7C2 HVR- GTYDGGFEY H3 25 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY 7C2.v2.2.LA.QQKPGQPPKL LIKYASILES GVPDRFSGSG SGTDFTLTIS (hu7C2) kappaSLQAEDVAVY YCQHSWEIPP WTFGQGTKVE IKRTVAAPSV light chainFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 26Hu7C2 IgG1 EVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA heavy chainPGQGLEWIGM IHPLDAEIRA NQKFRDRVTI TVDTSTSTAYLELSSLRSED TAVYYCARGT YDGGFEYWGQ GTLVTVSSASTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APTEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 27 Hu7C2.MIHPMDSEIRANQKFRD v2.1.S53M HVR-H2 28 Hu7C2. MIHPLDSEIRANQKFRD v2.1.S53LHVR-H2 29 Hu7C2. GTYDGGFKY v2.1.E101K HVR-H3 30 HumanizedDIVMTQSPDS LAVSLGERAT INCRASQSVS GSRFTYMHWY 7C2.v2.2.LA.QQKPGQPPKL LIKYASILES GVPDRFSGSG SGTDFTLTIS (Hu7C2)SLQAEDVAVY YCQHSWEIPP WTFGQGTKVE IKRTVAAPSV K149C kappaFIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWCVDNALQ light chainSGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 31Hu7C2 A118C EVQLVQSGAE VKKPGASVKV SCKASGYSFT GYWMNWVRQA IgG1 heavyPGQGLEWIGM IHPLDAEIRA NQKFRDRVTI TVDTSTSTAY chainLELSSLRSED TAVYYCARGT YDGGFEYWGQ GTLVTVSSCSTKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWNSGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYICNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 98 exemplaryMELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE human HER2THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV precursorQGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG protein, withDPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ signalLCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK sequenceGSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQCAAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFESMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQEVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSANIQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVFETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGAYSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTVPWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHCWGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHCLPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARCPSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDKGCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIKRRQQKIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETELRKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTSPKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQLMPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVRLVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHADGGKVPIKWMA LESILRRRFT HQSDVWSYGV TVWELMTFGAKPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWMIDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPLDSTFYRSLLE DDDMGDLVDA EEYLVTQQGF FCPDPAPGAGGMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEGAGSDVFDGDL GMGAKKGLQS LPTHDPSPLQ RYSEDPTVPLPSETDGYVAP LTCSPQPEYV NQPDVRPQPP SPREGPLPAARPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQGGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPVAnti-MUC16 Antibodies

In certain embodiments, ADCs comprise anti-MUC16 antibodies.

In some embodiments, described herein are ADCs comprising an anti-MUC16antibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 33 and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 34.

In one aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VH HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 35; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 35; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 37.

In another aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 32; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34. Inone embodiment, the antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 32; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 33; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 34.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 35, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 36, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 37; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 32, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 33, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 34.

In another aspect, described herein are ADCs comprising an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 32; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 33; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 34.

In any of the above embodiments, an anti-MUC16 antibody of an ADC ishumanized. In one embodiment, an anti-MUC16 antibody comprises HVRs asin any of the above embodiments, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework.

In another aspect, an anti-MUC16 antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 39. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO: 39 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-MUC16 antibodycomprising that sequence retains the ability to bind to MUC16. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 39. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 39. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-MUC16 antibody comprisesthe VH sequence of SEQ ID NO: 39, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35, (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 37.

In another aspect, an anti-MUC16 antibody of an ADC is provided, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 920/%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 38. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence ofSEQ ID NO:38 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-MUC16 antibody comprising that sequence retains the ability to bindto MUC16. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 38. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 38. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-MUC16 antibody comprisesthe VL sequence of SEQ ID NO: 38, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 32; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 33; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 34.

In another aspect, an ADC comprising an anti-MUC16 antibody is provided,wherein the antibody comprises a VH as in any of the embodimentsprovided above, and a VL as in any of the embodiments provided above.

In one embodiment, an ADC is provided, wherein the antibody comprisesthe VH and VL sequences in SEQ ID NO: 39 and SEQ ID NO: 38,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-MUC16 antibody provided herein. Forexample, in certain embodiments, an ADC is provided comprising anantibody that binds to the same epitope as an anti-MUC16 antibodycomprising a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ IDNO: 38, respectively.

In a further aspect, an anti-MUC16 antibody of an ADC according to anyof the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-MUC16 antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein.

TABLE 6 MUC16 Antibody Sequences. SEQ ID NO: Description Sequence 32Anti-Muc16 KASDLIHNWL A antibody HVR-L1 33 Anti-Muc16 YGATSLET antibodyHVR-L2 34 Anti-Muc16 QQYWTTPFT antibody HVR-L3 35 Anti-Muc16GYSITNDYAW N antibody HVR-H1 36 Anti-Muc16 GYISYSGYTT YNPSLKS antibodyHVR-H2 37 Anti-Muc16 ARWASGLDY antibody HVR-H3 38 Anti-Muc16DIQMTQSPSS LSASVGDRVT ITCKASDLIH antibody lightNWLAWYQQKP GKAPKLLIYG ATSLETGVPS chain variableRFSGSGSGTD FTLTISSLQP EDFATYYCQQ region YWTTPFTFGQ GTKVEIKR 39Anti-Muc16 EVQLVESGGG LVQPGGSLRL SCAASGYSIT antibody heavyNDYAWNWVRQ ARGKGLEWVG YISYSGYTTY chain variableNPSLKSRFTI SRDTSKNTLY LQMNSLRAED region TAVYYCARWA SGLDYWGQGT LVTVSSAnti-STEAP-1 Antibodies

In certain embodiments, ADCs comprise anti-STEAP-1 antibodies.

In some embodiments, described herein are ADCs comprising ananti-STEAP-1 antibody comprising at least one, two, three, four, five,or six HVRs selected from (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQID NO: 41; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:42; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

In one aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VH HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 40; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 40; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 41; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 42.

In another aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 43; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45. Inone embodiment, the antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 43; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 44; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 45.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 40, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 41, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 42; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 43, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 44, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 45.

In another aspect, described herein are ADCs comprising an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 45.

In any of the above embodiments, an anti-STEAP-1 antibody of an ADC ishumanized. In one embodiment, an anti-STEAP-1 antibody comprises HVRs asin any of the above embodiments, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework.

In another aspect, an anti-STEAP-1 antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 46. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO: 46 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-STEAP-1antibody comprising that sequence retains the ability to bind toSTEAP-1. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 46. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 46. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-STEAP-1 antibody comprisesthe VH sequence of SEQ ID NO: 46, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 40, (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 41, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 42.

In another aspect, an anti-STEAP-1 antibody of an ADC is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 47. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence ofSEQ ID NO: 47 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-STEAP-1 antibody comprising that sequence retains the ability tobind to STEAP-1. In certain embodiments, a total of 1 to 10 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 47 Incertain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 47. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theanti-STEAP-1 antibody comprises the VL sequence of SEQ ID NO: 47,including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

In another aspect, an ADC comprising an anti-STEAP-1 antibody isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above.

In one embodiment, an ADC is provided, wherein the antibody comprisesthe VH and VL sequences in SEQ ID NO: 46 and SEQ ID NO: 47,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-STEAP-1 antibody provided herein.For example, in certain embodiments, an ADC is provided comprising anantibody that binds to the same epitope as an anti-STEAP-1 antibodycomprising a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ IDNO: 47, respectively.

In a further aspect, an anti-STEAP-1 antibody of an ADC according to anyof the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-STEAP-1 antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein.

TABLE 7 STEAP Antibody Sequences. SEQ ID NO: Description Sequence 40Anti-STEAP-1 GYSITSDYAW N HVR-H1 41 Anti-STEAP-1 GYISNSGSTS YNPSLKSHVR-H2 42 Anti-STEAP-1 ERNYDYDDYY YAMDY HVR-H3 43 Anti-STEAP-1KSSQSLLYRS NQKNYLA HVR-L1 44 Anti-STEAP-1 WASTRES HVR-L2 45 Anti-STEAP1QQYYNYPRT HVR-L3 46 Anti-STEAP1 EVQLVESGGG LVQPGGSLRL SCAVSGYSITheavy chain SDYAWNWVRQ APGKGLEWVG YISNSGSTSY variable regionNPSLKSRFTI SRDTSKNTLY LQMNSLRAED TAVYYCARER NYDYDDYYYA MDYWGQGTLV TVSS47 Anti-STEAP1 DIQMTQSPSS LSASVGDRVT ITCKSSQSLL light chainYRSNQKNYLA WYQQKPGKAP LKKIYWASTR variable regionESGVPSRFSG SGSGTDFTLT ISSLQPEDFA TYYCQQYYNY PRTFGQGTKV EIKAnti-NaPi2b Antibodies

In certain embodiments, an ADC comprises anti-NaPi2b antibodies.

In some embodiments, described herein are ADCs comprising an anti-NaPi2bantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52 and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53.

In one aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VH HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 48; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 48; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 49; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 50.

In another aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 51; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53. Inone embodiment, the antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 51; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 52; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 53.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 48, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 49, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 50; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 51, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 52, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 53.

In another aspect, described herein are ADCs comprising an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 51; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53.

In any of the above embodiments, an anti-NaPi2b antibody of an ADC ishumanized. In one embodiment, an anti-NaPi2b antibody comprises HVRs asin any of the above embodiments, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework.

In another aspect, an anti-NaPi2b antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 990%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 54. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO: 54 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-NaPi2bantibody comprising that sequence retains the ability to bind to NaPi2b.In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 54. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 54. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-NaPi2b antibody comprisesthe VH sequence of SEQ ID NO: 54, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 48, (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 49, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 50.

In another aspect, an anti-NaPi2b antibody of an ADC is provided,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 930%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 55. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence ofSEQ ID NO: 55 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-NaPi2b antibody comprising that sequence retains the ability tobind to anti-NaPi2b. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in SEQ ID NO: 55.In certain embodiments, a total of 1 to 5 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 55. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-NaPi2bantibody comprises the VL sequence of SEQ ID NO: 55, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 52; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 53.

In another aspect, an ADC comprising an anti-NaPi2b antibody isprovided, wherein the antibody comprises a VH as in any of theembodiments provided above, and a VL as in any of the embodimentsprovided above.

In one embodiment, an ADC is provided, wherein the antibody comprisesthe VH and VL sequences in SEQ ID NO: 54 and SEQ ID NO: 55,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-NaPi2b antibody provided herein. Forexample, in certain embodiments, an ADC is provided comprising anantibody that binds to the same epitope as an anti-NaPi2b antibodycomprising a VH sequence of SEQ ID NO: 54 and a VL sequence of SEQ IDNO: 55, respectively.

In a further aspect, an anti-NaPi2b antibody of an ADC according to anyof the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-NaPi2b antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein.

TABLE 8 NaPi2B Antibody Sequences. SEQ ID NO: Description Sequence 48Anti-NaPi2b GFSGSDFAMS HVR-H1 10H1.11.4B 49 Anti-NaPi2bATIGRVAFHTYYPDSMKG HVR-H2 10H1.11.4B 50 Anti-NaPi2b ARHRGFDVGHFDF HVR-H310H1.11.4B 51 Anti-NaPi2b RSSETLVHSSGNTYLE HVR-L1 10H1.11.4B 52Anti-NaPi2b RVSNRFS HVR-L2 53 Anti-NaPi2b FQGSFNPLT HVR-L3 10H1.11.4B 54Anti-NaPi2b EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQ heavy chainAPGKGLEWVATIGRVAFHTYYPDSMKGRFTISRDNSKNT variable regionLYLQMNSLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVT 10H1.11.4B VSS V_(H) 55Anti-NaPi2b DIQMTQSPSSLSASVGDRVTITCRSSETLVHSSGNTYLE light chainWYQQKPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTL variable regionTISSLQPEDFATYYCFQGSFNPLTFGQGTKVEIKR 10H1.11.4B V_(L) 64 10H1.114BDIQMTQSPSSLSASVGDRVTITCRSSETLVHSSGNTYLE Light ChainWYQQKPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSFNPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 6510H1.114B EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQ Heavy ChainAPGKGLEWVATIGRVAFHTYYPDSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSKTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPFSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKAnti-CD79b Antibodies

In certain embodiments, ADCs comprise anti-CD79b antibodies.

In some embodiments, described herein are ADCs comprising an anti-CD79bantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 62; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 63.

In one aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VH HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 58; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 60.

In another aspect, described herein are ADCs comprising an antibody thatcomprises at least one, at least two, or all three VL HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 61; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 62;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 63. Inone embodiment, the antibody comprises (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 61; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 62; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 63.

In another aspect, an ADC comprises an antibody comprising (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 58, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 59, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 60; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 61, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 62, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 63.

In another aspect, described herein are ADCs comprising an antibody thatcomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:58; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 59; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 61; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 62; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 63.

In any of the above embodiments, an anti-CD79b antibody of an ADC ishumanized. In one embodiment, an anti-CD79b antibody comprises HVRs asin any of the above embodiments, and further comprises a human acceptorframework, e.g. a human immunoglobulin framework or a human consensusframework.

In another aspect, an anti-CD79b antibody of an ADC comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 990%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 56. In certain embodiments, a VH sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the amino acid sequence of SEQ ID NO: 56 containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-CD79b antibodycomprising that sequence retains the ability to bind to CD79b. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO: 56. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 56. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprisesthe VH sequence of SEQ ID NO: 8, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 58, (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 59, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 60.

In another aspect, an anti-CD79b antibody of an ADC is provided, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 57. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:57 contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Ly6E antibody comprising that sequence retains the ability to bindto CD79b. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 57. In certainembodiments, a total of 1 to 5 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 57. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprisesthe VL sequence of SEQ ID NO: 57, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 61; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 62; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 63.

In another aspect, described herein are ADCs comprising an anti-CD79bantibody is provided, wherein the antibody comprises a VH as in any ofthe embodiments provided above, and a VL as in any of the embodimentsprovided above.

In one embodiment, an ADC is provided, wherein the antibody comprisesthe VH and VL sequences in SEQ ID NO: 56 and SEQ ID NO: 57,respectively, including post-translational modifications of thosesequences.

In a further aspect, provided herein are ADCs comprising antibodies thatbind to the same epitope as an anti-CD79b antibody provided herein. Forexample, in certain embodiments, an ADC is provided comprising anantibody that binds to the same epitope as an anti-CD79b antibodycomprising a VH sequence of SEQ ID NO: 56 and a VL sequence of SEQ IDNO: 57, respectively.

In a further aspect, an anti-CD79b antibody of an ADC according to anyof the above embodiments is a monoclonal antibody, including a humanantibody. In one embodiment, an anti-CD79b antibody of an ADC is anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In another embodiment, the antibody is a substantially fulllength antibody, e.g., an IgG1 antibody, IgG2a antibody or otherantibody class or isotype as defined herein.

TABLE 9 CD79b Antibody Sequences. SEQ ID NO: Description Sequence 56anti-CD79b EVQLVESGGG LVQPGGSLRL SCAASGYTFS huMA79bv28SYWIEWVRQA PGKGLEWIGE ILPGGGDTNY heavy chainNEIFKGRATF SADTSKNTAY LQMNSLRAED variable regionTAVYYCTRRV PIRLDYWGQG TLVTVSS 57 anti-CD79bDIQLTQSPSS LSASVGDRVT ITCKASQSVD huMA79bv28YEGDSFLNWY QQKPGKAPKL LIYAASNLES light chainGVPSRFSGSG SGTDFTLTIS SLQPEDFATY variable regionYCQQSNEDPL TFGQGTKVEI KR 58 anti-CD79b GYTFSSYWIE huMA79bv28 HVR H1 59anti-CD79b GEILPGGGDTNYNEIFKG huMA79bv28 HVR H2 60 anti-CD79b TRRVPIRLDYhuMA79bv28 HVR H3 61 anti-CD79b KASQSVDYEGDSFLN huMA79bv28 HVR L1 62anti-CD79b AASNLES huMA79bv28 HVR L2 63 anti-CD79b QQSNEDPLT huMA79bv28HVR L3Anti-CD22 Antibodies

In certain embodiments, an ADC can comprise anti-CD22 antibodies, whichcomprise three light chain hypervariable regions (HVR-L1, HVR-L2 andHVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2 andHVR-H3). In one embodiment, the anti-CD22 antibody of an ADC comprisesthree light chain hypervariable regions and three heavy chainhypervariable regions (SEQ ID NO: 66-71), the sequences of which areshown below. In one embodiment, the anti-CD22 antibody of an ADCcomprises the variable light chain sequence of SEQ ID NO: 72 and thevariable heavy chain sequence of SEQ ID NO: 73. In one embodiment, theanti-CD22 antibody of ADCs of the present subject matter describedherein comprises the light chain sequence of SEQ ID NO: 74 and the heavychain sequence of SEQ ID NO: 75:

TABLE 10 Anti-CD22 Antibodies, h10F4.V3.K149C RSSQSIVHSVGNTFLESeq ID No: 66 HVR-L1 h10F4.V3.K149C KVSNRFS Seq ID No: 67 HVR-L2h10F4.V3.K149C FQGSQFPYT Seq ID No: 68 HVR-L3 h10F4.V3.K149C GYEFSRSWMNSeq ID No: 69 HVR-H1 h10F4.V3.K149C RIYPGDGDTNYSGKFKG Seq ID No: 70HVR-H2 h10F4.V3.K149C DGSSWDWYFDV Seq ID No: 71 HVR-H3 h10F4.V3.K149CDIQMTQSPSSLSASVGDRVTITCRSSQSIVHSVG Seq ID No: 72 V_(L)NTFLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCFQGSQFPYTFGQGTKVEIKR h10F4.V3.K149C EVQLVESGGGLVQPGGSLRLSCAASGYEFSRSWMSeq ID No: 73 V_(H) NWVRQAPGKGLEWVGRIYPGDGDTNYSGKFKGRFTISADTSKKTAYLQMNSLRAEDTAVYYCARDGSS WDWYFDVWGQGTLVTVSS h10F4.V3.K149CDIQMTQSPSSLSASVGDRVTITCRSSQSIVHSVG Seq ID No: 74 Light Chain NTFLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCFQGSQFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWCVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC h10F4.V3.K149CEVQLVESGGGLVQPGGSLRLSCAASGYEFSRSWM Seq ID No: 75 Heavy ChainNWVRQAPGKGLEWVGRIYPGDGDTNYSGKFKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCARDGSSWDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAnti-CD33 Antibodies

In certain embodiments, an ADC can comprise anti-CD33 antibodies, whichcomprise three light chain hypervariable regions and three heavy chainhypervariable regions, the sequences (SEQ ID NO:76-81) of which areshown below. In one embodiment, the anti-CD33 antibody of an ADCcomprises the variable light chain sequence of SEQ ID NO: 82 and thevariable heavy chain sequence of SEQ ID NO: 83.

TABLE 11 15G15.33- RSSQSLLHSNGYNYLD SEQ ID HVR L1 NO: 76 15G15.33-LGVNSVS SEQ ID HVR L2 NO: 77 15G15.33- MQALQTPWT SEQ ID HVR L3 NO: 7815G15.33- NHAIS SEQ ID HVR H1 NO: 79 15G-15.33- GIIPIFGTANYAQKFQG SEQ IDHVR H2 NO: 80 15G15.33- EWADVFDI SEQ ID HVR H3 NO: 81 15G15.33EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDW SEQ ID V_(L)YLQKPGQSPQLLIYLGVNSVSGVPDRFSGSGSGTDFTLKI NO: 82SRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK 15G15.33QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWVRQA SEQ ID V_(H)PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAF NO: 83MELSSLRSEDTAVYYCAREWADVFDIWGQGTMVTVSS

In one embodiment, the anti-CD33 antibody of an ADC comprises the lightchain sequence of SEQ ID NO: 84 and the heavy chain sequence of SEQ IDNO: 85. In one embodiment, the anti-CD33 antibody of an ADC comprisesthree light chain hypervariable regions and three heavy chainhypervariable regions, the sequences (Seq ID NO: 84-89) of which areshown below. In one embodiment, the anti-CD33 antibody of an ADCcomprises the variable light chain sequence of SEQ ID NO: 90 and thevariable heavy chain sequence of SEQ ID NO: 91. In one embodiment, theanti-CD33 antibody of ADC comprises the variable light chain sequence ofSEQ ID NO: 92 and the variable heavy chain sequence of SEQ ID NO: 93. Inone embodiment, the anti-CD33 antibody of ADCs of the present subjectmatter described herein comprises the variable light chain sequence ofSEQ ID NO: 94 and the variable heavy chain sequence of SEQ ID NO: 95. Inone embodiment, the anti-CD33 antibody of ADCs of the present subjectmatter described herein comprises the variable light chain sequence ofSEQ ID NO: 96 and the variable heavy chain sequence of SEQ ID NO: 97.

TABLE 12 9C3-HVR RASQGIRNDLG Seq ID NO: L1 84  9C3-HVR AASSLQSSeq ID NO: L2 85 9C3-HVR LQHNSYPWT Seq ID NO: L3 86 9C3-HVR GNYMSSeq ID NO: H1 87 9C3-HVR LIYSGDSTYYADSVKG Seq ID NO: H2 88 9C3-HVRDGYYVSDMVV Seq ID NO: H3 89 9C3 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ Seq ID NO:QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLT 90ISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK 9C3 V_(H)EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV Seq ID NO:RQAPGKGLEWVSLIYSGDSTYYADSVKGRFNISRDIS 91KNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKG TTVTVSS 9C3.2 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ Seq ID NO:QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLT 92ISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK 9C3.2 V_(H)EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV Seq ID NO:RQAPGKGLEWVSLIYSGDSTYYADSVKGRFTISRDIS 93KNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKG TTVTVSS 9C3.3 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ Seq ID NO:QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLT 94ISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK 9C3.3 V_(H)EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV Seq ID NO:RQAPGKGLEWVSLIYSGDSTYYADSVKGRFSISRDIS 95KNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKG TTVTVSS 9C3.4 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ Seq ID NO:QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLT 96ISSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK 9C3.4 V_(H)EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV Seq ID NO:RQAPGKGLEWVSLIYSGDSTYYADSVKGRFAISRDIS 97KNTVYLQMNSLRVEDTAVYYCVRDGYYVSDMVVWGKG TTVTVSSAntibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M. e.g., from 10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20®; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophotometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Linkers (L1)

As described herein, a “linker” (L1) is a bifunctional ormultifunctional moiety that can be used to link one or more drugmoieties (D) to an antibody (Ab) to form an ADC. In some embodiments,ADCs can be prepared using a L1 having reactive functionalities forcovalently attaching to the drug and to the antibody. For example, insome embodiments, a cysteine thiol of an antibody (Ab) can form a bondwith a reactive functional group of a linker or a linker L1-drugintermediate to make an ADC. Particularly, the chemical structure of thelinker can have significant impact on both the efficacy and the safetyof an ADC (Ducry & Stump, Bioconjugate Chem, 2010, 21, 5-13). Choosingthe right linker influences proper drug delivery to the intendedcellular compartment of target cells.

Linkers can be generally divided into two categories: cleavable (such aspeptide, hydrazone, or disulfide) or non-cleavable (such as thioether).Peptide linkers, such as Valine-Citrulline (Val-Cit) that can behydrolyzed by lysosomal enzymes (such as Cathepsin B) have been used toconnect the drug with the antibody (U.S. Pat. No. 6,214,345). They havebeen particularly useful, due in part to their relative stability insystemic circulation and the ability to efficiently release the drug intumor. However, the chemical space represented by natural peptides islimited; therefore, it is desirable to have a variety of non-peptidelinkers which act like peptides and can be effectively cleaved bylysosomal proteases. The greater diversity of non-peptide structures mayyield novel, beneficial properties that are not afforded by the peptidelinkers. Provided herein are different types of non-peptide linkers forlinker L1 that can be cleaved by lysosomal enzymes.

a. Peptidomimetic Linkers

Provided herein are different types of non-peptide, peptidomimeticlinkers for ADC's that are cleavable by lysosomal enzymes. For example,the amide bond in the middle of a dipeptide (e.g. Val-Cit) was replacedwith an amide mimic; and/or entire amino acid (e.g., valine amino acidin Val-Cit dipeptide) was replaced with a non-amino acid moiety (e.g.,cycloalkyl dicarbonyl structures (for example, ring size=4 or 5)).

When L1 is a peptidomimetic linker, it is represented by the followingformula-Str-(PM)-Sp-,wherein:Str is a stretcher unit covalently attached to Ab;Sp is a bond or spacer unit covalently attached to a biologically activemoiety;PM is a non-peptide chemical moiety selected from the group consistingof:

W is —NH-heterocycloalkyl- or heterocycloalkyl;Y is heteroaryl, aryl, —C(O)C₁-C₆ alkylene, C₁-C₆ alkylene-NH₂, C₁-C₆alkylene-NH—CH₃, C₁-C₆ alkylene-N—(CH₃)₂, C₁-C₆ alkenyl or C₁-C₆alkylenyl;each R⁶ is independently C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, (C₁-C₁₀alkyl)NHC(NH)NH₂ or (C₁-C₁₀ alkyl)NHC(O)NH₂;R⁷ and R⁸ are each independently H, C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl,arylalkyl or heteroaryl, or R⁷ and R⁸ together may form a C₃-C₇cycloalkyl;R⁹ and R¹⁰ are each independently C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl,arylalkyl, heteroaryl, (C₁-C₁₀ alkyl)OCH₂—, or R⁹ and R⁰ may form aC₃-C₇ cycloalkyl ring;

p has a value from about 1 to about 10;

In embodiments, Y is heteroaryl; R⁹ and R¹⁰ together form a cyclobutylring.

In embodiments, Y is a moiety selected from the group consisting of:

In embodiments, Str is a chemical moiety represented by the followingformula:

wherein R¹¹ is selected from the group consisting of C₁-C₁₀ alkylene.C₁-C₁₀ alkenyl. C₃-C₈ cycloalkyl, (C₁-C₈ alkylene)O—, and C₁-C₁₀alkylene-C(O)N(R^(a))—C₂-C₆ alkylene, where each alkylene may besubstituted by one to five substituents selected from the groupconsisting of halo, trifluoromethyl, difluoromethyl, amino, alkylamino,cyano, sulfonyl, sulfonamide, sulfoxide, hydroxy, alkoxy, ester,carboxylic acid, alkylthio, C₃-C₈ cycloalkyl, C₄-C₇ heterocycloalkyl,aryl, arylalkyl, and heteroaryl each R^(a) is independently H or C₁-C₆alkyl; Sp is —Ar—R^(b)—, wherein Ar is aryl or heteroaryl, R^(b) is(C₁-C₁₀ alkylene)O—.

In embodiments, Str has the formula:

wherein R¹² is selected from C₁-C₁₀ alkylene, C₁-C₁₀ alkenyl, (C₁-C₁₀alkylene)O—, N(R^(c))—(C₂-C₆ alkylene)-N(R^(c)) and N(R^(c))—(C₂-C₆alkylene); where each R^(C) is independently H or C₁-C₆ alkyl; Sp is—Ar—R^(b)—, wherein Ar is aryl or heteroaryl, R^(b) is (C₁-C₁₀alkylene)O— or Sp-C₁-C₆ alkylene-C(O)NH—.

In embodiments, L is a non-peptide chemical moiety represented by thefollowing formula

R⁶ is C₁-C₆ alkyl, C₁-C₆ alkenyl, (C₁-C₆ alkyl)NHC(NH)NH₂ or (C₁-C₆alkyl)NHC(O)NH₂;R⁷ and R⁸ are each independently H or C₁-C₁₀ alkyl.

In embodiments, L is non-peptide chemical moiety represented by thefollowing formula

R⁶ is C₁-C₆ alkyl, (C₁-C₆ alkyl)NHC(NH)NH₂ or (C₁-C₆ alkyl)NHC(O)NH₂;

R⁹ and R¹⁰ together form a C₃-C₇ cycloalkyl ring.

In embodiments, L is non-peptide chemical moiety represented by thefollowing formula

R⁶ is C₁-C₆ alkyl, (C₁-C₆ alkyl)NHC(NH)NH₂ or (C₁-C₆ alkyl)NHC(O)NH₂.

In some embodiments, the linker may be a peptidomimetic linker such asthose described in WO2015/095227, WO2015/095124 or WO2015/095223, whichdocuments are hereby incorporated by reference in their entirety.

b. Non-Peptidomimetic Linkers

In an aspect, a Linker L1 forms a disulfide bond with the antibody, andthe linker has the structure:

wherein R¹, R², R³, and R⁴ are independently selected from the groupconsisting of H, optionally substituted branched or linear C₁-C₅ alkyl,and optionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring. The linker is covalently bound to an antibody and a drug asfollows:

In another aspect, a linker L1 has a functionality that is capable ofreacting with an antibody having a cysteine with a free thiol present toform a covalent bond. Nonlimiting exemplary such reactivefunctionalities include maleimide, haloacetamides, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See,e.g., the conjugation method at page 766 of Klussman, et al (2004),Bioconjugate Chemistry 15(4):765-773, and the Examples herein.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”). Various linker components are knownin the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug.Nonlimiting exemplary cleavable linkers include acid-labile linkers(e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020).

In certain embodiments, a linker has the following Formula:-A_(a)W_(w)—Y_(y)—

wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W isan “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacerunit”, and y is 0, 1, or 2. Exemplary embodiments of such linkers aredescribed in U.S. Pat. No. 7,498,298, which is expressly incorporatedherein by reference.

In some embodiments, a linker component comprises a “stretcher unit”that links an antibody to another linker component or to a biologicallyactive moiety. Nonlimiting exemplary stretcher units are shown below(wherein the wavy line indicates sites of covalent attachment to anantibody, biologically active, or additional linker components):

3. Drug (D)

As used herein, the term “drug” refers to a biologically active moleculecomprising a secondary nitrogen-containing heteroaryl. The term“secondary nitrogen” refers to a nitrogen that is available for covalentfunctionalization. That is, the drug has a secondary nitrogen-containingheteroaryl group wherein said nitrogen is capable of forming a covalentbond to a linker (L1) as further defined herein. The secondary nitrogencan be present in the drug within any portion of the drug molecule,whether part of a core functionality that has been highly substituted ora pendant piece, which may or may not be substituted. For example, drugsmay contain heteroaryl groups such as indoles, indazoles,benzimidazoles, benzotriazoles, pyrroles, pyrrolopyridines, imidazoles,triazoles, tetrazoles, and the like. Specific examples include, but arenot limited to the following heteroaryl structures:

The heteroaryl groups may also exist in alternate tautomeric forms, ascan be seen in such non-limiting examples as:

The aforementioned heteroaryl groups may be substituted in many forms,such as, for example, additional functionalization or fused rings. Thesecondary nitrogen can be represented in a structure as N—H. It is to beunderstood that this means that in the ADC, the N—H refers to the pointof attachment of the drug:

in the ADC.

As used herein, the term “biologically active molecule” refers to asmall molecule capable of performing a function or an action orstimulating or responding to a function in a biological context, e.g.,an organism, a cell, an in vitro model, or in vivo systems. Inparticular, a small molecule capable of treating a disease or conditionin a subject. The function may be related to a receptor, enzyme, ionchannel, target, or the like.

Useful drugs can include cytotoxic agents that inhibit or prevent acellular function and/or causes cell death or destruction; growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; antitumor oranticancer agents, such as alkylating agents; alkyl sulfonates;aziridines; ethylenimines and methylamelamines; acetogenins (especiallybullatacin and bullatacinone); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin aberrant cell proliferation; topoisomerase 1 inhibitors; proteosomeinhibitors; EGFR inhibitors; tyrosine kinase inhibitors;serine-threonine kinase inhibitors; farnesyltransferase inhibitors; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Additional drugs include “anti-hormonal agents” or “endocrinetherapeutics” which act to regulate, reduce, block, or inhibit theeffects of hormones that can promote the growth of cancer. They may behormones themselves, including, but not limited to: anti-estrogens withmixed agonist/antagonist profile; selective estrogen receptor modulators(SERMs); pure anti-estrogens without agonist properties; aromataseinhibitors, including steroidal aromatase inhibitors and nonsteroidalaromatase inhibitors; lutenizing hormone-releaseing hormone agonists;sex steroids; estrogens; and androgens/retinoids; estrogen receptordown-regulators (ERDs); anti-androgens; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe mammal being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens; non-steroidal anti-inflammatorydrugs (NSAIDs); anti-inflammatory agents; cyclooxygenase inhibitors,leukotriene receptor antagonists; purine antagonists; steroids;dihydrofolate reductase inhibitors; anti-malarial agents

Non-limiting examples of drugs comprising a secondary nitrogencontaining heteroaryl are amanitin, vinblastine, vincristine,duocarmycin A, duocarmycin SA, CC-1065, adozelesin, U-76074, U-73073,carzelesin (U-80244), KW-2189, diazonamide A, esomeprazole,aripiprazole, valsartan, lansoprazole, rabeprazole, pometrexed,olmesartan, tadalafil, pantoprazole, candosartan, omeprazole, sunitinib,pemetrexed, alectinib, dacarbazine, semaxanib, dacinostat, dovitinib,mebendazole, and pimobendan. Examples of drugs also include thefollowing compounds:

Additionally, a person of ordinary skill in the art would recognize thatmany more examples of biologically active molecules comprising anitrogen-containing heteroaryl exist that have not received formalnames, but still have undergone testing which would place said moleculesin the category as defined herein.

The subject matter described herein also includes pharmaceuticallyacceptable salts of antibody-drug conjugates of Formula I.

If the antibody-drug conjugate of Formula I is cationic, or has afunctional group which may be cationic (e.g. NH₂ may be NH₃ ⁺), thedesired pharmaceutically acceptable salt may be prepared by any suitablemethod available in the art, for example, treatment of the free basewith an inorganic acid, such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid andthe like, or with an organic acid, such as acetic acid, maleic acid,succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid,oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the antibody-drug conjugate of Formula I is anionic, or has afunctional group which may be anionic (e.g. —COOH may be —COO⁻), thedesired pharmaceutically acceptable salt may be prepared by any suitablemethod, for example, treatment of the free acid with an inorganic ororganic base, such as an amine (primary, secondary or tertiary), analkali metal hydroxide or alkaline earth metal hydroxide, or the like.Illustrative examples of suitable salts include, but are not limited to,organic salts derived from amino acids, such as glycine and arginine,ammonia, primary, secondary, and tertiary amines, and cyclic amines,such as piperidine, morpholine and piperazine, and inorganic saltsderived from sodium, calcium, potassium, magnesium, manganese, iron,copper, zinc, aluminum and lithium.

Illustrative examples of other suitable salts include, but are notlimited to, organic salts derived from amino acids, such as glycine andarginine, ammonia, primary, secondary, and tertiary amines, and cyclicamines, such as piperidine, morpholine and piperazine, and inorganicsalts derived from sodium, calcium, potassium, magnesium, manganese,iron, copper, zinc, aluminum and lithium.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1. Analytical LCMS Conditions

Condition A

Experiments were performed on a SHIMADZU 20A HPLC (with a PDA detector)with a SHIMADZU 2010EV MSD mass spectrometer using ESI as the ionizationsource. The LC separation was conducted using a MK RP18e 25-2 mm columnwith a 1.5 mL/min flow rate. Solvent A was 1.5 mL TFA per 4 L water andsolvent B was 0.75 mL TFA per 4 L acetonitrile. The gradient consistedof 5-95% solvent B over 0.7 min followed by holding at 95% B for 0.4min, followed by equilibration for 0.4 min. LC column temperature was50° C. UV absorbance was monitored at 220 nm and 254 nm, and mass specfull scan was applied to all experiments.

Condition B

Experiments were performed on an Agilent 1290 UHPLC coupled with anAgilent MSD (6140) mass spectrometer using ESI as the ionization source.The LC separation was conducted using a Phenomenex XB-C18, 1.7 um,50×2.1 mm column with a 0.4 mL/min flow rate. Solvent A was water with0.1% formic acid and solvent B was acetonitrile with 0.1% formic acid.The gradient consisted of 2-98% solvent B over 7 min followed by holdingat 98% B for 1.5 min, followed by equilibration for 1.5 min. LC columntemperature was 40° C. UV absorbance was monitored at 220 nm and 254 nm,and mass spec full scan was applied to all experiments.

Condition C

Experiments were performed on a Waters Acquity UPLC with a Waters LCTPremier XE mass spectrometer using ESI ionization. The LC separation wasconducted using an Acquity UPLC BEH C18, 1.7 um, 2.1×50 mm column and a0.6 mL/min flow rate. Solvent A was water with 0.05% TFA and solvent Bwas acetonitrile with 0.05% TFA. The gradient consisted of 2-98% solventB over 5 min followed by holding at 98% B for 0.5 min followed byequilibration for 0.5 min. LC column temperature was 40° C. UVabsorbance was monitored at 220 nm and 254 nm, and mass spec full scanwas applied to all experiments.

Example 2. Synthesis of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-9-(((R)-2-((5-nitropyridin-2-yl)disulfaneyl)propoxy)carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (Compound 8)

Step 1:(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

To a solution of compound 1 (prepared according to: J. Med. Chem. 2015,58, 8128; 500 mg, 1.1 mmol) in MeOH (100 mL)/water (25 mL) was added aNaOH solution in water (3.0 M, 9.0 mL, 27 mmol). After the mixture wasstirred at 25° C. for 18 h, it was concentrated to about 5.0 mL, pouredinto 1.0 M HCl (10 mL), and extracted with EtOAc (3×50.0 mL). Thecombined organic layers were dried over Na₂SO₄ and concentrated to givethe crude 2 (400 mg, 83%) as a yellow solid. This material was useddirectly in the next step.

Step 2: tert-butyl(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

To a solution of compound 2 (400.0 mg, 0.90 mmol) in DCM (5.0 mL) wasadded tert-butyl 2,2,2-trichloroacetimidate (15 mL, 83.89 mmol). Themixture was stirred at 25° C. for 60 h, washed with water (3×50.0 mL),brine (50.0 mL), and dried over Na₂SO₄. The organic layer wasconcentrated and purified by column chromatography (10% EtOAc inpetroleum ether) to give 3 (310 mg, 69%) as a yellow solid.

Step 3: (R)-2-(tritylthio)propan-1-ol

TFA (0.40 mL, 5.23 mmol) was added to a solution of compound 4 (preparedas described in: WO 2013055987; 482.0 mg, 5.23 mmol) andtriphenylmethanol (953 mg, 3.66 mmol) in CHCl₃ (25 mL). The mixture wasstirred at 25° C. for 5 h and then diluted with DCM (25 mL). Theresulting solution was washed with sat. NaHCO₃ (50.0 mL), dried overNa₂SO₄, and concentrated under reduced pressure. The residue waspurified by flash column chromatography (20% EtOAc in petroleum ether,Rf=0.5) to give compound 5 (600 mg, 34%) as a yellow oil.

Step 4: (R)-2-(tritylthio)propyl carbonochloridate

To a solution of compound 5 (600 mg, 1.79 mmol) in DCM (5.0 mL) wasadded diphosgene (497 mg, 2.51 mmol) over 2 min at 0° C. DIEA (232 mg,1.79 mmol) was then added over 1 min. The mixture was stirred at 0° C.for 1 h and then at 25° C. for 2.0 h. The reaction mixture wasconcentrated and dissolved in THF (1.0 mL). Heptanes were then addeduntil a white solid appeared. This solid was removed by filtrationthrough celite. The filtrate was concentrated under reduced pressure togive compound 6 (710 mg, 99.7%) as a white solid. This material was useddirectly in next step.

Step 5: (R)-2-(tritylthio)propyl(1R,3R)-1-(4-((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2,6-difluorophenyl)-2-(2-fluoro-2-methylpropyl)-3-methyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-9-carboxylate

To a solution of compound 3 (140.0 mg, 0.280 mmol) in DMF (2.0 mL) wasadded sodium hydride (60%, 7.41 mg, 0.310 mmol) at 0° C. The resultingmixture was stirred at 25° C. for 30 min and a solution of compound 6(111 mg, 0.280 mmol) in DMF (1.0 mL) was then added. After the mixturewas stirred at 25° C. for 1.0 h, it was concentrated under reducedpressure and the residue purified by column chromatography (10% EtOAc inpetroleum ether, Rf=0.6) to afford compound 7 (92 mg, 38%). LCMS(Condition A): R_(T)=1.12 min, m/z=859.3 [M+H]⁺.

Step 6:(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-9-(((R)-2-((5-nitropyridin-2-yl)disulfaneyl)propoxy)carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

TFA (0.30 mL) was added to a solution of compound 7 (80.0 mg, 0.090mmol), triisopropylsilane (16.2 mg, 0.10 mmol), and2,2′-dithiobis(5-nitropyridine) (86.7 mg, 0.280 mmol) in DCM (1.0 mL).After the mixture was stirred at 25° C. for 18 h, toluene (40.0 mL) wasadded. The mixture was concentrated and the residue purified by columnchromatography (50% EtOAc in petroleum ether) to afford compound 8 (27.2mg, 37%) as a yellow solid. LCMS (Condition A): R_(T)=0.92 min,m/z=715.0 [M+Na]⁺; ¹H NMR (400 MHz, CDCl₃) δ 9.12 (s, 1H), 8.10-8.04 (m,2H), 7.70-7.68 (d, J=8.8 Hz, 1H), 7.51-7.42 (m, 2H), 7.24-7.23 (m, 3H),7.12-6.89 (m, 1H), 6.32-6.28 (d, J=15.9 Hz, 1H), 5.76 (br, 1H),4.22-4.18 (m, 2H), 3.27-3.15 (m, 1H), 2.67-2.45 (m, 4H), 2.22-2.11 (m,1H), 1.41-1.36 (m, 3H), 1.19-1.07 (m, 3H), 0.89-0.81 (m, 3H).

Example 3. Synthesis of(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-9-((2-((5-nitropyridin-2-yl)disulfaneyl)ethoxy)carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid (Compound 13)

Step 1: 2-(tritylthio)ethan-1-ol

TFA (2.2 mL, 28 mmol) was added to a solution of compound 9 (2.0 mL,28.5 mmol) and triphenylmethanol (5.2 g, 20 mmol) in CHCl₃ (100 mL). Themixture was stirred at 25° C. for 4 h and then diluted with DCM (75 mL).The resulting solution was washed with sat. NaHCO₃ (200 mL), dried overMgSO₄, filtered, and concentrated under reduced pressure. The residuewas purified by flash column chromatography (0-40% iPrOAc in heptanes)to give compound 10 (2.1 g, 33%) as a white solid. ¹H NMR (400 MHz,CDCl₃) δ 7.47-7.38 (m, 5H), 7.32-7.15 (m, 10H), 3.41 (q, J=6.2 Hz, 2H),2.48 (t, J=6.2 Hz, 2H), 1.48 (t, J=6.1 Hz, 1H).

Step 2: 2-(tritylthio)ethyl carbonochloridate

To a solution of compound 10 (1.5 mg, 4.7 mmol) in DCM (30 mL) was addeddiphosgene (1.3 g, 6.6 mmol) over 2 min at 0° C. DIEA (0.82 mL, 4.7mmol) was then added over 1 min. The mixture was stirred at 0° C. for 1h and then at 25° C. for 3 h. The reaction mixture was concentrated andredissolved in THF (10 mL). Heptanes were added until a white solidappeared (approximately 30 mL). This solid was removed by filtrationthrough celite. The filtrate was concentrated under reduced pressure togive crude compound 11 as a yellow oil. This material was used directlyin the next step.

Step 3: 2-(tritylthio)ethyl(1R,3R)-1-(4-((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)-2,6-difluorophenyl)-2-(2-fluoro-2-methylpropyl)-3-methyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-9-carboxylate

Sodium hydride (60%, 61 mg, 1.53 mmol) was added to a solution ofcompound 3 (693 mg, 1.39 mmol) in DMF (25 mL) at 0° C. The resultingmixture was stirred at 25° C. for 30 min and a solution of compound 11(532 mg, 1.39 mmol) in DMF (5.0 mL) was then added. After the mixturewas stirred at 25° C. for 10 min, the volatiles were removed underreduced pressure. The residue was partitioned between iPrOAc (2×150 mL)and 0.5 M HCl (150 mL). The combined organic layers were dried overMgSO₄, filtered, and concentrated under reduced pressure. The residuewas purified by column chromatography (0-20% iPrOAc in heptanes) toafford compound 12 (189 mg, 16%) as a white foam. ¹H NMR (400 MHz,CDCl₃) δ 8.14 (d, J=7.5 Hz, 1H), 7.49-7.13 (m, 11H), 6.79 (d, J=10.5 Hz,2H), 6.23 (d, J=15.9 Hz, 1H), 5.74-5.70 (m, 1H), 4.99 (hept, J=6.3 Hz,1H), 3.86 (t, J=7.0 Hz, 1H), 3.84-3.76 (m, 1H), 3.72-3.62 (m, 1H),3.36-3.24 (m, 1H), 2.68 (dd, J=16.7, 4.6 Hz, 1H), 2.63-2.42 (m, 6H),2.36-2.27 (m, 1H), 1.54 (s, 3H), 1.53 (s, 9H), 1.44 (d, J=21.7 Hz, 3H),1.27 (d, J=21.1 Hz, 3H), 1.12 (d, J=6.7 Hz, 3H).

Step 4:(E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-9-((2-((5-nitropyridin-2-yl)disulfaneyl)ethoxy)carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylicacid

TFA (0.30 mL) was added to a solution of compound 12 (18 mg, 0.021mmol), triisopropylsilane (16.2 mg, 0.10 mmol), and2,2′-dithiobis(5-nitropyridine) (20 mg, 0.064 mmol) in DCM (1.0 mL).After the mixture was stirred at 25° C. for 16 h, toluene (40.0 mL) wasadded. The mixture was concentrated and the residue purified by reversephase HPLC (Column=Gemini-NX C18 Sum, 110A, 50×30 mm, temp=25° C.;Eluents: A=0.1% formic acid in water, B=acetonitrile; 50-90% B over 10min, flow=60 mL/min; detection=270 nM) to afford compound 13 (7.0 mg,50%) as an off-white solid. LCMS (Condition B): R_(T)=7.24 min,m/z=701.2 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 9.13 (d, J=2.6 Hz, 1H),8.36 (dd, J=8.8, 2.7 Hz, 1H), 8.09-7.99 (m, 1H), 7.91 (d, J=8.9 Hz, 1H),7.59-7.50 (m, 1H), 7.43 (d, J=16.0 Hz, 1H), 7.33-7.25 (m, 3H), 6.58 (d,J=16.0 Hz, 1H), 5.75 (s, 1H), 4.50-4.35 (m, 2H), 3.23-3.03 (m, 5H),2.80-2.52 (m, 4H), 1.39 (d, J=21.5 Hz, 3H), 1.26 (d, J=21.1 Hz, 3H),1.10 (d, J=6.7 Hz, 3H).

Example 4. Synthesis of1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)2-methyl 6-(benzyloxy)-1H-indole-1,2-dicarboxylate (Compound 17)

Step 1: 2-methyl 1-(4-nitrophenyl)6-(benzyloxy)-1H-indole-1,2-dicarboxylate

Sodium hydride (60%, 235 mg, 5.88 mmol) was added to a solution ofcompound 14 (1.74 g, 6.19 mmol) in THF (50 mL) at 0° C. The resultingmixture was stirred at 25° C. for 2.5 h and a solution of4-nitro-phenylchloroformate (1.22 g, 5.88 mmol) in THF (15 mL) wasadded. After the mixture was stirred at 25° C. for 4.0 h, it wasfiltered through Celite. The filtrate was concentrated under reducedpressure to afford a yellow oil. This material was dissolved in amixture of Et₂O (100 mL) and heptanes (40 mL) and the resulting solutionwas concentrated under reduced pressure until a solid appeared(approximately 80 mL volume). The solid was collected by vacuumfiltration and air-dried overnight to give crude 15 (1.22 g) that wascontaminated with a significant amount (ca. 33%) of an unknown impurity.This material was used in the next step without further purification.

Step 2:1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)2-methyl 6-(benzyloxy)-1H-indole-1,2-dicarboxylate

A solution of crude compound 15 (135 mg, approx 0.252 mmol), di-peptide16 (prepared as described in: WO 2015162293 and WO 2015162293; 0.028 g,0.036 mmol), and DMAP (0.030 g, 0.246 mmol) in DMF (10 mL) was stirredat 25° C. for 5 days. The volatiles were then removed under reducedpressure and the residue purified by preparative HPLC (Column=Gemini-NXC18 5 um, 110A, 50×30 mm, temp=25° C.; Eluents: A=0.1% ammoniumhydroxide in water, B=acetonitrile; 40-80% B over 10 min, flow=60mL/min; detection=254 nM) to give compound 17 (0.028 g, 14%) as anoff-white solid. LCMS (Condition B): R_(T)=6.22 min, m/z=787.4 [M+H]⁺;¹H NMR (400 MHz, DMSO-d) δ 10.13 (s, 1H), 7.99 (d, 0.1=7.7 Hz, 1H),7.69-7.59 (m, 3H), 7.54 (d, J=2.3 Hz, 1H), 7.48-7.30 (m, 7H), 7.28 (d,J=0.8 Hz, 1H), 7.04 (dd, J=8.7, 2.3 Hz, 1H), 6.74 (d, J=8.9 Hz, 1H),5.95 (t, J=5.9 Hz, 1H), 5.40 (br s, 2H), 5.36 (s, 2H), 5.09 (s, 2H),4.50-4.41 (m, 1H), 3.87-3.81 (m, 1H), 3.71 (s, 3H), 3.07-2.88 (m, 2H),2.01-1.90 (m, 1H), 1.73-1.52 (m, 2H), 1.38 (s, 9H), 0.86 (d, J=6.8 Hz,3H), 0.81 (d, J=6.8 Hz, 3H).

Example 5. Synthesis of 2-methyl 1-(2-(tritylthio)ethyl)6-(benzyloxy)-1H-indole-1,2-dicarboxylate (Compound 18)

Sodium hydride (60%, 78 mg, 1.96 mmol) was added to a solution ofcompound 14 (500 mg, 1.78 mmol) in DMF (15 mL) at 0° C. The resultingmixture was stirred at 25° C. for 45 min and a solution of compound 11(681 mg, 1.78 mmol) in DMF (5.0 mL) was then added. After the mixturewas stirred at 25° C. for 1.0 h, it was partitioned between iPrOAc(2×150 mL) and 0.5 M HCl (150 mL). The combined organic layers weredried over MgSO₄, filtered, and concentrated under reduced pressure. Theresidue was purified by column chromatography (0-30% iPrOAc in heptanes)to afford compound 18 (440 mg, 39%) as a white foam. LCMS (Condition C):R_(T)=5.26 min, m/z=628.1 [M+H]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 7.63 (dd,J=8.7, 0.4 Hz, 1H), 7.56 (dt, J=2.2, 0.6 Hz, 1H), 7.44-7.20 (m, 21H),7.05 (dd, J=8.7, 2.3 Hz, 1H), 5.15 (s, 2H), 4.13 (t, J=6.3 Hz, 2H), 3.75(s, 3H), 2.57 (t, J=6.3 Hz, 2H).

Example 6. Synthesis of Antibody-Drug Conjugates

Anti-HER2 7C2 LC K149C and anti-B7H4 1D11 LC K149C were conjugated tocompound 8 or compound 13 via the engineered LC K149C cysteine residues.Solutions of a given antibody at 10 mg/mL in 50 mM Tris pH 8.5 werereduced with 50 molar excess of DTT at ambient temperature for 16-18hours. The reduced antibody was purified using SP HP cationic exchangechromatography. The purified antibody in 50 mM Tris pH 8 was re-oxidizedusing 15 molar excess of DHAA dissolved in DMA at ambient temperaturefor 2-3 hours. The antibody was again purified using SP HP cationicexchange column to remove DHAA and aggregates. Three to five-fold molarequivalents of compound 8 or 13 in DMF were added to a 5 to 10 mg/mLsolution of the purified antibody in 100 mM Tris pH 8.5-9.0 followed byadditional DMF for a final concentration of 10% DMF. The couplingreaction was then incubated at ambient temperature for 3 to 4 hours. Theconjugated antibody was purified using either of the following methods:cationic exchange chromatography using HiTrap SP HP resin or by Zebaspin 7 kDa MWCO desalting resin followed by removal of excess free drugusing dextran coated charcoal. The resulting purified conjugate wasformulated using dialysis with 10 kDa MWCO Slide-a-Lyzer dialysiscassette into 20 mM histidine acetate pH 5.5, 240 mM sucrose, 0.02%polysorbate-20.

Conjugates produced using the conjugation reaction conditions and eitherof the purification schemes typically afforded protein yields of 50 to80%. All of the conjugates were characterized in regards to aggregation(SEC HPLC), drug to antibody ratio (LC/MS), percent of nitro-pyridyldisulfide (nitroPDS) species conjugated (LC/MS) and amount of free drug(LC/MS) present in the final conjugates. In all of the conjugates, <5%of the conjugated species to the antibody were nitroPDS resulting fromconjugation to the nitroPDS sulfur rather than the sulfur on the drugside of the disulfide bond. Detailed characterization data for eachconjugate are provided in Table 13 below.

TABLE 13 CNJ Linker Free drug lot Antibody Drug DAR nitroPDS Aggregationcontent Yield CNJ-1 Anti-HER2 7C2 LC Compound 1.9 4.9% 1.5% <5% 55%K149C 13 CNJ-2 Anti-B7H4 1D11v1.9 Compound 1.9 1.9% 3.9% <5% 52% varD LCK149C 13 CNJ-3 Anti-HER2 7C2 LC Compound 2.0 0.5% 0.5% <2% 70% K149C  8CNJ-4 Anti-B7H4 1D11v1.9 Compound 2.0 0.6% 0.6% <2% 77% varD LC K149C  8

Example 7. Biological Assays

Whole Blood Sample Preparation

Stability samples were generated in mouse (CB17 SCID), rat(Sprague-Dawley), cynomologus monkey and human whole blood plasma aswell as buffer (0 and 24 h timepoints). Blood was collected bybioreclamation, then shipped cold overnight, and samples were createdimmediately on arrival. To create stability samples, initial dilutionsof the source conjugates were made in buffer (1×PBS, 0.5% BSA, 15 ppmproclin) so that all molecules were 1 mg/mL in concentration. Then a1:10× dilution (36 uL of 1 mg/mL initial dilution+324 uL blood orbuffer) was performed to generate the stability samples with a finalcompound concentration of 100 ug/mL. Once mixed, 150 μL of the wholeblood/buffer stability samples was aliquoted into two separate sets oftubes for the two different time points. The 0 h time points were thenplaced in a −80° C. freezer, while the 24 h time points were placed on ashaker in a 37° C. incubator. When the 24 h samples reached the giventime point they were also placed in the −80° C. freezer.

Affinity-Capture LC-MS Assays for Stability Determination of Whole BloodSamples

The whole blood stability samples were evaluated using anaffinity-capture LC-MS assay. First, streptavidin-coated magnetic beads(Life Technologies Corporation, Grand Island, N.Y.) were washed 2× withHBS-EP buffer (GE Healthcare, Sunnyvale, Calif.), then mixed withbiotinylated HER2 anti-idiotypic antibody using the KingFisher Flex(Thermo Fisher Scientific, Waltham, Mass.) and incubated for 2 h at roomtemperature with gentle agitation. After 2 h, the SA-bead/biotin-xId Abcomplex was washed 2× with HBS-EP buffer, mixed with the diluted wholeblood stability samples and then incubated for 2 h at room temperaturewith gentle agitation. After 2 h, the SA-bead/biotin-xId Ab/samplecomplex was washed 2× with HBS-EP buffer, mixed with the deglycosylationenzyme PNGase F (New England BioLabs, Ipswich, Mass.) and incubatedovernight at 37° C. with gentle agitation. After the overnightincubation, the deglycosylated SA-bead/biotin-xId Ab/sample complex waswashed 2× with HBS-EP buffer, followed by 2× washes with water (OptimaH₂O, Fisher Scientific, Pittsburgh, Pa.) and finally a 1× wash with 10%acetonitrile. The beads were placed in 300% acetonitrile/0.1% formicacid for elution where they were incubated for 30 min at roomtemperature with gentle agitation before the beads were collected. Theeluted samples were injected and loaded onto a Thermo ScientificPepSwift RP monolithic column (500 μm×5 cm) maintained at 65° C. Thesamples were separated on the column using a Waters Acquity UPLC systemat a flow rate of 20 μL/min with the following gradient: 20% B (95%acetonitrile+0.1% formic acid) at 0-2 min; 35% B at 2.5 min; 65% B at 5min; 95% B at 5.5 min; 5% B at 6 min. The column was directly coupledfor online detection with a Waters Synapt G2-S Q-ToF mass spectrometryoperated in positive ESI with an acquisition mass range from 500 to 5000Th (m/z).

Breast Cancer Cell ERα High Content Fluorescence Imaging Assay (F10)

MCF7-neo/HER2 breast cancer cells were seeded on day 1 at a density of10,000 cells per well in 384 well poly-lysine coated tissue cultureplate (Greiner #T-3101-4), in 50 uL/well RPMI (phenol red free), 10% FBS(Charcoal stripped), containing L-glutamine. On day-2, AntibodyConjugates were thawed at RT and were each diluted to 60 ug/mL in 37° C.growth media, followed by a 20-point 2× serial dilution across a 384well plate (Ref: 781091). 10 uL of each sample from the serial dilutionwas transferred to the wells of the cell plates. The highest workingconcentration of the ADCs was 10 ug/mL. Cell plate columns 1, 2, 23 and24 were left untreated for data normalization while Columns 3-22contained the ADC dilutions. After compound treatment, cell plates werestored in a 37° C. incubator for 72 h. Fixation and permeabilizationwere carried out using a Biotek EL406 plate washer and dispenser onday-5 as follows. Cells were fixed by addition of 15 uL of 16%paraformaldehyde (Electron Microscopy Sciences #15710-S) directly to the50 uL cell culture medium in each well using the peristaltic pump 5 uLcassette on a Biotek EL406 (final concentration of formaldehyde was3.7%/c w/v). Samples were incubated 30 minutes. Well contents wereaspirated and 50 uL/well of Phosphate Buffered Saline (PBS) containing0.5% w/v bovine serum albumen and 0.5% v/v Triton X-100 (AntibodyDilution Buffer) were added to each well. Samples were incubated for 30minutes. The well contents were aspirated and washed 3 times with 100uL/well of PBS. Immunofluorescence staining of estrogen receptor alpha(ESR1) was carried out using a Biotek EL406 plate washer and dispenseras follows. The well supernatant was aspirated from the wells and 25uL/well of anti-ESR1 mAb (F10) (Santa Cruz sc-8002) diluted 1:1000 inAntibody Dilution Buffer was dispensed. Samples were incubated for 2hours at room temperature and then washed 4 times with 100 uL/well ofPBS. 25 uL/well of secondary antibody solution (Alexafluor 488 conjugateanti-mouse IgG (LifeTechnologies #A21202) diluted 1:1000 and Hoechst33342 1 ug/mL diluted in Antibody Dilution Buffer) were dispensed intoeach well. The samples were incubated for 2 hours at room temperatureand then washed 3 times with 100 uL/well of PBS using a Biotek EL406.Quantitative fluorescence imaging of ESR1 was carried out using aCellomics Arrayscan V (Thermo). Fluorescence images of the samples[Channel 1: XF53 Hoechst (DNA stain); Channel 2: XF53 FITC (ESR1 stain)]were acquired using a Cellomics VTI Arrayscan using the Bioapplication“Compartmental Analysis” using the auto-exposure (based on DMSO controlwells) setting “peak target percentile” set to 25% target saturation forboth channels. Channel 1 (DNA stain) was used to define the nuclearregion (Circ). Measurements of “Mean_CircAvgIntCh2”, which is theAlexafluor 488 fluorescence intensity (ESR1) within the nuclear region,was measured on a per cell basis and averaged over all the measuredcells. Data analysis was carried out using GraphPad Prism 6, with DMSOand no primary antibody control treated samples being used to define the0% and 100% changes in ESR1. The dose-response log(inhibitor) vs.response was used to define the inflexion point of curve (EC₅₀) and theplateau of the maximal effect.

In Vivo Modulation of ERα Levels in MCF7-Neo/HER2 Derived Tumors

Human breast cancer MCF7 cells were originally obtained from AmericanType Culture Collection (Rockville, Md.) and were engineered atGenentech to overexpress HER2 to generate MCF7-neo/HER2. Four days priorto tumor cell implantation, 55 female NCR nude mice (Taconic) were puton ad libitum estrogen fortified water containing 0.8 ug/mL1713-estradiol (Sigma; St. Louis, Mo.) in 0.003% ethanol. MCF7-neo/HER2cells, resuspended in 50% phenol red-free Matrigel (Becton DickinsonBioscience; San Jose, Calif.) and Hank's Balanced Salt Solution, wereinoculated subcutaneously in the number ⅔ mammary fat pad. Each mousewas injected with 5×106 cells. Tumors were monitored until they reachedan approximate tumor volume of 400 mm³. Seventeen days after tumorimplantation (four days prior to dosing conjugates), mice weredistributed into five groups of 4 mice per group and estrogen fortifiedwater was removed and replaced with ad libitum regular drinking water.Twenty-one days after tumor implantation, designated as Day 1 of thestudy, mice were administered a single intravenous injection of vehicle(20 mM histidine acetate pH 5.5, 240 mM sucrose, 0.02% polysorbate 20),CNJ-1 (HER2) at 2, 10, or 25 mg/kg or CNJ-2 (B7H4) at 25 mg/kg in avolume of 200 uL. Twenty-four hours post-dose blood was collected viaretro-orbital bleeds and processed for serum. On Day 4, all mice wereeuthanized and tumors and blood were collected. Tumors were flash frozenat −80° C. and blood was processed for serum.

For protein extraction, each frozen tumor was transferred to atissueTUBE (Covaris) and cryofractured using a cryoPREP Impactor set toimpact level 5 (Covaris). Half of the pulverized tumor samples wereresuspended in 300 uL cell extraction buffer (FNN0011, LifeTechnologies) supplemented with protease inhibitor cocktail (Roche),phosphatase inhibitor cocktail (Sigma) and containing one ⅛″ Coneball(Glenmills) and two 3 mm Zirconia beads (Glenmills). The samples werehomogenized in a Geno Grinder (SPEX Sample Prep) for 1.5 min at 1,500rpm, and the extracts were cleared twice by centrifugation (14,000 rpmat 4° C. for 10 min). Protein concentrations were determined by BCAassay (Thermo Fisher Scientific). For each tumor sample, 26 μg of totalprotein was separated on a 4-12% NuPAGE Bis-Tris gel using MOPS buffer(Thermo Fisher Scientific) and transferred to a nitrocellulose membrane.The membranes were blocked for 1 h in Odyssey blocking buffer (LI-COR,cat #927-40000) and incubated overnight with primary antibodies directedagainst ERα (dilution 1:1000, Novus Biologicals, cat. #NBP2-26481) andβ-tubulin (dilution 1:1000, LI-COR, cat. #926-42212). Following three 15min washes in PBS-tween, the membranes were incubated with the secondaryantibodies IR Dye 800 CW Donkey anti Rabbit (dilution 1:10,000, LI-COR,cat #926-32213) and IR Dye 680 RD Donkey anti Mouse (dilution 1:10,000,LI-COR, cat #926-68072) for 45 min. After three 15 min washes inPBS-tween, the membranes were scanned using an Odyssey CLx instrument(LI-COR) and the ERα and tubulin levels quantified. The ERα/tubulinratio and standard error of the mean (SEM) were calculated using Exceland plotted using Prism6 (Graphpad).

Efforts have been made to ensure accuracy with respect to numbers used(e.g. amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepracticing the subject matter described herein. The present disclosureis in no way limited to just the methods and materials described.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this subject matter belongs, and are consistent with:Singleton et al (1994) Dictionary of Microbiology and Molecular Biology,2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York.

Throughout this specification and the claims, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. It is understood that embodimentsdescribed herein include “consisting of” and/or “consisting essentiallyof” embodiments.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments ±50%, in some embodiments±20%, in some embodiments ±10%, in some embodiments ±5%, in someembodiments ±1%, in some embodiments ±0.5%, and in some embodiments±0.1% from the specified amount, as such variations are appropriate toperform the disclosed methods or employ the disclosed compositions.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of the range and any other stated or intervening value in thatstated range, is encompassed. The upper and lower limits of these smallranges which may independently be included in the smaller rangers isalso encompassed, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

Many modifications and other embodiments set forth herein will come tomind to one skilled in the art to which this subject matter pertainshaving the benefit of the teachings presented in the foregoingdescriptions and the associated drawings. Therefore, it is to beunderstood that the subject matter is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

That which is claimed:
 1. A method for preparing an antibody-drugconjugate of Formula I:Ab-(L1-D)p  I or a pharmaceutically acceptable salt thereof, wherein Abis an antibody; L1 is a linking moiety with the structure

wherein, R¹, R², R³, and R⁴ are independently selected from the groupconsisting of H, optionally substituted branched or linear C₁-C₅ alkyl,and optionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylwherein said optionally substituted alkyl or cycloalkyl may besubstituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl, nitrile,halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy, alkylthio, sulfonate,amino, alkylamino, acylamino, carbamoyl, alkylcarbamoyl, or nitro; D is

wherein L1 is covalently bonded to the secondary nitrogen; and p is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10; the method comprising: i. contacting acompound of Formula IIT-L1-D  II with an antibody, wherein, L1 and D are as described above, Tis a leaving group having the structure R⁵—S, wherein R⁵ is anoptionally substituted pyridine, wherein an antibody-drug conjugate ofFormula I is prepared.
 2. The method of claim 1, wherein T-L1-D has thefollowing formula:

wherein, R⁵ is selected from the group consisting of unsubstitutedpyridine and nitropyridine.
 3. The method of claim 1, wherein saidantibody-drug conjugate of Formula I has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 4. The method of claim 1,wherein said antibody-drug conjugate of Formula I has the structure:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 5. The method of claim 1,wherein the antibody-drug conjugate of Formula I is selected from thegroup consisting of:

wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 6. A method for preparinga compound of Formula III:

wherein, R¹, R², R³, and R⁴ are independently selected from the groupconsisting of H, optionally substituted branched or linear C₁-C₅ alkyl,and optionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring wherein said optionally substituted alkyl or cycloalkyl may besubstituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl, nitrile,halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy, alkylthio, sulfonate,amino, alkylamino, acylamino, carbamoyl, alkylcarbamoyl, or nitro; and Dis

wherein the carbonyl in Formula III is covalently bonded to thesecondary nitrogen in D; R⁵ is selected from the group consisting ofoptionally substituted pyridine and nitropyridine; the methodcomprising: i. contacting a compound of Formula V:

wherein LG is a leaving group and PG is a protecting group, with acompound, D, to prepare a compound of Formula VI:

and, ii. deprotecting the compound of Formula VI under acidic conditionsto prepare a compound of Formula III:


7. A compound of Formula III:

wherein, R¹, R², R³, and R⁴ are independently selected from the groupconsisting of H, optionally substituted branched or linear C₁-C₅ alkyl,and optionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring wherein said optionally substituted alkyl or cycloalkyl may besubstituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl, nitrile,halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy, alkylthio, sulfonate,amino, alkylamino, acylamino, carbamoyl, alkylcarbamoyl, or nitro; R⁵ isselected from the group consisting of optionally substituted pyridineand nitropyridine; and D is

wherein the carbonyl in Formula III is covalently bonded to thesecondary nitrogen in D.
 8. The method of claim 1 for preparing anantibody-drug conjugate of Formula IV:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; R¹, R², R³, and R⁴ are independently selected from the groupconsisting of H, optionally substituted branched or linear C₁-C₅ alkyl,and optionally substituted C₃-C₆ cycloalkyl, or R³ and R⁴ taken togetherwith the carbon atom to which they are bound form a C₃-C₆ cycloalkylring, wherein said optionally substituted alkyl or cycloalkyl may besubstituted with alkyl, cycloalkyl, aryl, heteroaryl, hydroxyl, nitrile,halo, alkoxy, haloalkoxy, arylalkoxy, acyloxy, alkylthio, sulfonate,amino, alkylamino, acylamino, carbamoyl, alkylcarbamoyl, or nitro; D is

wherein the carbonyl in Formula IV is covalently bonded to the secondarynitrogen in D; and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; the methodcomprising: i. contacting a compound of Formula V:

with a compound D, wherein LG is a leaving group and PG is a protectinggroup, to prepare a compound of Formula VI:

ii. contacting the compound of Formula VI with a disulfide R⁵—S—S—R⁵under acidic conditions to prepare a compound of Formula III:

wherein R⁵ is an optionally substituted pyridine; and iii. contacting acompound of Formula III with an antibody to prepare an antibody-drugconjugate of Formula IV.
 9. The method of claim 2 wherein the compoundof Formula III is selected from:


10. The method of claim 1 wherein the antibody is Anti-HER2 orAnti-B7H4.